Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PROTEIN KINASE INHIBITORS AND METHODS FOR USING THEREOF
Cross-Reference to Related Applications
[0001] This application claims the benefit of U.S. provisional application
serial number
60/944,457, filed June 15, 2007, which is incorporated herein by reference in
its entirety.
Technical Field
[0002] The invention relates to protein kinase inhibitors, and methods of
using such
compounds.
Background Art
[0003] The protein kinases include a large number of family members, which
play a central
role in regulating a wide variety of cellular function. A partial, non-
limiting, list of these kinases
include: receptor tyrosine kinases such as platelet derived growth factor
receptor (PDGFR), nerve
growth factor receptorTrkB, C-Met, and fibroblast growth factor receptor(FGFR-
3); non-receptor
tyrosine kinases such as Abl and the corresponding fusion kinase Bcr-Abl, Lck,
Csk, Fes, Bmx
and Src; and serine/threonine kinases such as B-Raf, C-Raf, Syk, MAP kinases
(e.g., MKK4,
MKK6, etc.) and SAPK2a, SAPK20 and SAPK3. Aberrant kinase activity has been
observed in
many disease states including benign and malignant proliferative disorders, as
well as diseases
resulting from inappropriate activation of the immune and nervous systems.
Therefore, inhibition
of these kinases would have multiple therapeutic indications.
Disclosure of the Invention
[0004] The invention provides compounds and pharmaceutical compositions
thereof, which
may be useful as protein kinase inhibitors.
[0005] In one aspect, the invention provides compounds having Formula (1):
1
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N-0~\N
R1 ~ II
R4" v\J (R2)m
/N~ L1 ~
N ll \ /~ 2- - 3
~N L Y (R )n
R5 (1)
or pharmaceutically acceptable salts thereof, wherein:
Ll is NR, NRCO or NRS01_2;
LZ are independently NRCO, NRCONR, CONR, NRSO1_2 or SO1_2NR;
Y is a C3_7 cycloalkyl, C3_7 heterocycloalkyl, or a monocyclic or fused 5-10
membered
aryl or heteroaryl containing N, 0 or S;
Rl and R5 are independently H, an optionally halogenated C1_6 alkyl, NR2 or
halo;
R2 is an optionally halogenated CI-6 alkyl or halo;
R3 is halo, substituted or unsubstituted CI-6 alkyl, C2_6 alkenyl, or C2_6
alkynyl; optionally
halogenated C1_6 alkoxy, XR8, XO(CR2)pR9, O(CR2)pNR6R7, XNR6R7 or
XNR(CR2)pNR6R7;
R4 is NR6R7 , NR(CR2)pNR6R7, NRCONR6R7 or NRCOZR6;
R6 and R7 are independently H, an optionally halogenated CI-6 alkyl, C2_6
alkenyl or C2_6
alkynyl; C1_6 alkanol, XR8 or XO(CR2)pR9; or R6 and R7 together with N in
NR6R7 may form an
optionally substituted ring;
R8 and R9 are independently an optionally substituted C3_7 cycloalkyl, 5-7
membered
aryl, heterocyclic or heteroaryl; or R9 is H;
each R is H or CI-6 alkyl;
each X is a bond or a Cl_4 alkylene;
m is 0-2; and
n and p are independently 0-4.
[0006] In the above Fonnula (1), Ll may be NH. In other examples, L 2 is NHCO,
CONH, or
NHCONH. In yet other examples, Rl may be H. In yet other examples, R 2 is CH3.
[0007] In another embodiment, the compound is of Formula (2) or (3):
2
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NN
RH
N N N
NH-Y-(R3)n
~N
R5 0 (2) or
NN
~~J H
R4 , N N
N~ N NH Y-(R3)n
~
R5 O (3).
[0008] In the above Formula (1), (2) or (3), Y may be a monocyclic or fused 5-
10 membered
aryl or heteroaryl containing N, 0 or S. For example, Y may be phenyl,
pyridyl, thienyl,
pyrazolyl, isoxazolyl, furanyl or pyrrolyl. In other examples, R3 is halo,
C1_6 alkyl optionally
substituted with halo, hydroxyl, alkoxy, or cyano; optionally halogenated C1_6
alkoxy, XR8,
XO(CR2)pR9, O(CR2)pNR6R', XNR6R' or XNR(CR2)pNR6R7. For example, R6 and R7
together
with N form an optionally substituted piperidinyl, morpholinyl, piperazinyl,
pyrrolidinyl,
pyrrolidonyl or imidazolyl. In other examples, R8 and R9 are independently an
optionally
substituted C3_7 cycloalkyl, piperidinyl, morpholinyl, piperazinyl,
pyrrolidinyl, pyrrolidonyl,
imidazolyl, pyridyl, phenyl, furanyl, naphthalenyl, pyrimidinyl, triazolyl,
isothiazolyl, isoxazolyl,
pyrazolyl or pyrazinyl.
[0009] In the above Formula (1), (2) or (3), each optionally substituted ring
is optionally
substituted with halo, optionally halogenated Cl_6 alkyl, C2_6 alkenyl, or
C2_6 alkynyl; nitro, cyano,
XCO1_2R10, XO(CR2)pR10, XS(CR2)pR10, XR8, XNR'O(CR2)pR10, XNR(CRz)pNRz,
XNRCOR10
XNRCONR2, XNR(CRz)pOR, XNR(C=NR)NR2, XCONR10(CR2)pR10, XCONR(CRz)pNRz,
XNSOi_zR, XNRSR, XSOi_zRB, XSOi_2NR10(CR2)pR10 or XSNR2; wherein R10 is H,
optionally
halogenated C1_6 alkyl, C3_7 cycloalkyl, 5-7 membered aryl, heterocyclic or
heteroaryl.
[0010] In another aspect, the present invention provides pharmaceutical
compositions
comprising a compound having Formula (1), (2) or (3), and a pharmaceutically
acceptable
excipient.
[0011] The invention also provides methods for modulating a protein kinase,
comprising
administering to a system or a subject in need thereof, a therapeutically
effective amount of a
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compound having Formula (1), (2) or (3), or pharmaceutically acceptable salts
or pharmaceutical
compositions thereof, thereby modulating said protein kinase. In one
embodiment, the invention
provides methods for inhibiting a kinase, comprising administering to a system
or a subject in
need thereof, a therapeutically effective amount of a compound of Formula (1),
(2) or (3).
[0012] Examples of protein kinases which may be modulated or inhibited using
the
compounds of the invention include but are not limited to Alk, Abl, Aurora-A,
B-Raf, C-Raf,
Bcr-Abl, BRK, Blk, Bmx, BTK, C-Kit, C-Src, EphBl, EphB2, EphB4, FLT1, Fms,
F1t3, Fyn,
FRK3, JAK2, KDR, Lck, Lyn, PDGFRa, PDGFR(3, PKCa, p38, Src, SIK, Syk, Tie2 and
TrkB
kinases. More particularly, the compounds of Formula (1), (2) or (3) may be
used for inhibiting
B-Raf.
[0013] In yet another aspect, the invention provides methods for ameliorating
or treating a
condition mediated by a protein kinase, such as a B-Raf-mediated condition,
comprising
administering to a system or subject in need of such treatment an effective
amount of a compound
having Formula (1), (2) or (3), or pharmaceutically acceptable salts or
pharmaceutical
compositions thereof, and optionally in combination with a second therapeutic
agent, thereby
treating said condition. For example, the compounds of the invention,
optionally in combination
with a chemotherapeutic agent, may be used to treat a cell proliferative
disorder, including but not
limited to, melanoma, leukemia, chronic myelogenous leukemia, lymphoma,
osteosarcoma, or
breast, renal, prostate, colorectal, thyroid, ovarian, pancreatic, neuronal,
lung, uterine or
gastrointestinal tumor. The compounds of the invention may also be used to
treat an autoimmune
disorder, including but not limited to systemic lupus erythematosus,
inflammatory bowel disease,
rheumatoid arthritis, or multiple scleriosis.
[0014] In the above methods for using the compounds of the invention, a
compound having
Formula (1), (2) or (3) may be administered to a system comprising cells or
tissues. In other
embodiments, a compound having Formula (1), (2) or (3) may be administered to
a human or
animal subject.
[0015] The invention also provides for the use of a compound of Formula (1),
(2) or (3) in the
manufacture of a medicament for treating a condition mediated by a protein
kinase.
Definitions
[0016] "Alkyl" refers to a moiety and as a structural element of other groups,
for example
halo-substituted alkyl and alkoxy, and may be straight-chained or branched. An
optionally
substituted alkyl, alkenyl or alkynyl as used herein may be optionally
halogenated (e.g., CF3), or
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may have one or more carbons that is substituted or replaced with a
heteroatom, such as NR, 0 or
S (e.g., -OCHZCHZO-, alkylthiol, thioalkoxy, alkylamine, etc).
[0017] "Aryl" refers to a monocyclic or fused bicyclic aromatic ring
containing carbon atoms.
For example, aryl may be phenyl or naphthyl. "Arylene" means a divalent
radical derived from
an aryl group.
[0018] "Heteroaryl" as used herein is as defined for aryl above, where one or
more of the ring
members are a heteroatom. Examples of heteroaryls include but are not limited
to pyridyl,
indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl,
benzothiopyranyl,
benzo[1,3]dioxolyl, imidazolyl, benzoimidazolyl, pyrimidinyl, furanyl,
oxazolyl, isoxazolyl,
triazolyl, tetrazolyl, pyrazolyl, thienyl, etc.
[0019] A "carbocyclic ring" as used herein refers to a saturated or partially
unsaturated,
monocyclic, fused bicyclic or bridged polycyclic ring containing carbon atoms,
which may
optionally be substituted, for example, with =0. Examples of carbocyclic rings
include but are
not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cyclopropylene, cyclohexanone,
etc.
[0020] A "heterocyclic ring" as used herein is as defined for a carbocyclic
ring above,
wherein one or more ring carbons is a heteroatom. For example, a heterocyclic
ring may contain
N, O, S, -N=, -S-, -S(O)9 -S(O)Z-, or -NR- wherein R may be hydrogen, Cl4alkyl
or a protecting
group. Examples of heterocyclic rings include but are not limited to
morpholinyl, pyrrolidinyl,
pyrrolidin-2-one, piperazinyl, piperidinyl, piperidinone, 1,4-dioxa-8-aza-
spiro[4.5]dec-8-yl, etc.
[0021] The terms "co-administration" or "combined administration" or the like
as used herein
are meant to encompass administration of the selected therapeutic agents to a
single patient, and
are intended to include treatment regimens in which the agents are not
necessarily administered
by the same route of administration or at the same time.
[0022] The term "pharmaceutical combination" as used herein refers to a
product obtained
from mixing or combining active ingredients, and includes both fixed and non-
fixed
combinations of the active ingredients. The term "fixed combination" means
that the active
ingredients, e.g. a compound of Formula (1) and a co-agent, are both
administered to a patient
simultaneously in the form of a single entity or dosage. The term "non-fixed
combination" means
that the active ingredients, e.g. a compound of Formula (1) and a co-agent,
are both administered
to a patient as separate entities either simultaneously or sequentially with
no specific time limits,
wherein such administration provides therapeutically effective levels of the
active ingredients in
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the body of the patient. The latter also applies to cocktail therapy, e.g. the
administration of three
or more active ingredients.
[0023] The term "therapeutically effective amount" means the amount of the
subject
compound that will elicit a biological or medical response in a cell, tissue,
organ, system, animal
or human that is being sought by the researcher, veterinarian, medical doctor
or other clinician.
[0024] The term "administration" or "administering" of the subject compound
means
providing a compound of the invention and prodrugs thereof to a subject in
need of treatment.
[0025] "Kinase Panel" is a list of kinases including but not limited to Abl,
JAK2, JAK3,
ALK, JNK1a1, KDR, Aurora-A, Lck, Blk, MAPK1, Bmx, MAPKAP-K2, BRK, MEK1,
CaMKII, C-Met, CDK1/cyclinB, p70S6K, CHK2, PAK2, CK1, PDGFRa, CK2, PDK1, C-
Kit,
Pim-2, C-Raf, PKA, CSK, PKBa, Src, PKCa, DYRK2, P1k3, EGFR, ROCK-I, Fes, Ron,
FGFR-
3, Ros, F1t3, SAPK2a, Fms, SGK, Fyn, SIK, GSK30, Syk, IGFR, Tie-2, IKKB, TrkB,
IR,
WNK3, IRAK4, ZAP-70, ITK, AMPK, LIMK1, Rsk2, Axl, LKB1, SAPK20, BrSK2, Lyn,
SAPK3, BTK, MAPKAP-K3, SAPK4, CaMKIV, MARK1, Snk, CDK2/cyclinA, MINK, SRPK1,
CDK3/cyclinE, MKK4, TAK1, CDK5/p25, MKK6, TBK1, CDK6/cyclinD, MLCK, TrkA,
CDK7/cyclinH/MAT1, MRCK(3, TSSK1, CHK1, MSK1, Yes, CK1d, MST2, ZIPK, MuSK,
DAPK2, NEK2, DDR2, NEK6, DMPK, PAK4, DRAK1, PAR-1Ba, EphAl, PDGFR(3, EphA2,
Pim-1, EphA5, PKB(3, EphB2, PKC(3I, EphB4, PKCB, FGFR1, PKCrl, FGFR2, PKCO,
FGFR4,
PKD2, Fgr, PKG1(3, Fltl, PRK2, Hck, PYK2, HIPK2, Ret, IKKa, RIPK2, IRR, ROCK-
II,
JNK2a2, Rse, JNK3, Rskl(h), P13 Ky, P13 K8 and PI3-K(3.
Modes of Carrying Out the Invention
[0026] The present invention provides compounds and pharmaceutical
compositions thereof,
which may be useful as protein kinase inhibitors.
[0027] In one aspect, the invention provides compounds having Fonnula (1):
N~N
R1 ~ II
4
R v\J (R2)m
N~ L1 ~
N ll \ /\ 2- - 3
~N L Y (R )n
R5 (1)
or pharmaceutically acceptable salts thereof, wherein:
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Ll is NR, NRCO or NRS01_2;
LZ are independently NRCO, NRCONR, CONR, NRSO1_2 or SO1_2NR;
Y is a C3_7 cycloalkyl, C3_7 heterocycloalkyl, or a monocyclic or fused 5-10
membered
aryl or heteroaryl containing N, 0 or S;
Rl and R5 are independently H, an optionally halogenated CI-6 alkyl, NR2 or
halo;
R2 is an optionally halogenated CI-6 alkyl or halo;
R3 is halo, substituted or unsubstituted CI-6 alkyl, C2_6 alkenyl, or C2_6
alkynyl; optionally
halogenated C1_6 alkoxy, XR8, XO(CR2)pR9, O(CR2)pNR6R7, XNR6R7 or
XNR(CR2)pNR6R7;
R4 is NR6R7 , NR(CR2)pNR6R7, NRCONR6R7 or NRCOZR6;
R6 and R7 are independently H, an optionally halogenated CI-6 alkyl, C2_6
alkenyl or C2_6
alkynyl; C1_6 alkanol, XR8 or XO(CR2)pR9; or R6 and R7 together with N in
NR6R7 may form an
optionally substituted ring;
R8 and R9 are independently an optionally substituted C3_7 cycloalkyl, 5-7
membered
aryl, heterocyclic or heteroaryl; or R9 is H;
each R is H or CI-6 alkyl;
each X is a bond or a Cl_4 alkylene;
m is 0-2; and
n and p are independently 0-4.
[0028] In one embodiment, the compound is of Formula (2) or (3):
NN
RH -
// ~ i/ NH-Y-(R3)n
N N N
~N ~
R5 0 (2) or
NN
~
R4 \\~ H b// ~ I/ - a
N NH-]~Y (R )n
R5 O (3).
[0029] Representative compounds of the invention include but are not limited
to:
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N- {4-Methyl-3- [2-(6-methylamino-pyrimidin-4-yl)-2H- [ 1,2,4]triazol-3-
ylamino]-
phenyl } -3-trifluoromethyl-benzamide;
4-Methyl-3- [2-(6-methylamino-pyrimidin-4-yl)-2H- [ 1,2,4]triazol-3-ylamino]-N-
(3-
trifluoromethyl-phenyl)-benzamide;
4-Methyl-3-(3-methyl-l-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)-
N-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)benzamide;
1- { 4-Methyl-3-[2-(6-methylamino-pyrimidin-4-yl)-2H-[ 1,2,4]triazol-3-
ylamino]-
phenyl } -3-(3-pyrrolidin-1-ylmethyl-5-trifluoromethyl-phenyl)-urea;
3-(1,1-difluoroethyl)-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)benzamide;
2-tert-butyl-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-
5-
ylamino)phenyl)isonicotinamide;
2-(2-hydroxypropan-2-yl)-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)isonicotinamide;
2-(1,1-difluoroethyl)-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)isonicotinamide;
3-(4-ethylpiperazin-1-yl)-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)-5 -(trifluoromethyl)benzamide;
3-isopropoxy-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-
5-
ylamino)phenyl)benzamide;
N-(4-methyl-3 -(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-
3 -(trifluoromethoxy)benzamide;
3-(2-cyanobutan-2-yl)-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)benzamide;
3-(2-cyanopropan-2-yl)-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)benzamide;
3-(2-hydroxypropan-2-yl)-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)benzamide;
N-(4-methyl-3 -(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-
3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)benzamide;
N-(4-methyl-3 -(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-
3-(piperazin-1-yl)-5-(trifluoromethyl)benzamide;
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N-(4-methyl-3 -(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-
3 -(1-methylpiperidin-4-yloxy) -5 - (trifluoromethyl)benzamide;
3-(4-(2-hydroxyethyl)piperazin-1-yl)-N-(4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-
yl)-1H-1,2,4-triazol-5-ylamino)phenyl)-5-(trifluoromethyl)benzamide;
N-(4-methyl-3 -(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-
3 -(piperidin-4-yloxy)-5-(trifluoromethyl)benzamide;
N-(3-(1-(6-(2,3-dihydroxypropylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)-4-
methylphenyl)-3-(trifluoromethyl)benzamide;
4-fluoro-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-3-(trifluoromethyl)benzamide;
3-fluoro-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-5-(trifluoromethyl)benzamide;
4-chloro-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-3-(trifluoromethyl)benzamide;
4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-N-(3-
(4-
methylpiperazin-l-yl)-5-(trifluoromethyl)phenyl)benzamide;
N-(3-tert-butylphenyl)-4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-
triazol-
-yl amino)benzamide;
3-(1-(6-(cyclopropylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-4-methyl-
N-(3-
(4-methylpiperazin-l-yl) -5 -(trifluoromethyl)phenyl)benzamide;
3-(1-(6-(cyclopropylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-N-(3-(4-
ethylpiperazin-l-yl)-5-(trifluoromethyl)phenyl)-4-methylbenzamide;
N-(3-(4-ethylpiperazin-l-yl)-5-(trifluoromethyl)phenyl)-4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
3-(1-(6-aminopyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-4-methyl-N-(3-(4-
methylpiperazin-l-yl)-5-(trifluoromethyl)phenyl)benzamide;
3-(1-(6-(methoxyamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-4-methyl-N-
(3-(4-
methylpiperazin-l-yl)-5-(trifluoromethyl)phenyl)benzamide;
N-(3-(4-ethylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)-3-(1-(6-
(methoxyamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-4-methylbenzamide;
3-(1-(6-aminopyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-N-(3-(4-
ethylpiperazin-1-yl)-
5 -(trifluoromethyl)phenyl) -4-methylbenzamide;
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3-(1-(6-(cyclopropylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-N-(3-(4-
isopropylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)-4-methylbenzamide;
N-(3-(4-isopropylpiperazin-l-yl)-5-(trifluoromethyl)phenyl)-4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
N-(3-(4-isopropylpiperazin-l-yl)-5-(trifluoromethyl)phenyl)-3-(1-(6-
(methoxyamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-4-methylbenzamide;
N-(2-tert-butylpyridin-4-yl)-4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)benzamide;
4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-N-(4-
(trifluoromethyl)pyridin-2-yl)benzamide;
N-(3-(3-hydroxycyclobutyl)-5-(trifluoromethyl)phenyl)-4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
2-methoxy-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-6-(trifluoromethyl)isonicotinamide;
4-methyl-3-(3-methyl-l-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)-
N-(3-(trifluoromethyl)phenyl)benzamide;
N-(3-(4-hydroxypiperidin-1-yl)-5-(trifluoromethyl)phenyl)-4-methyl-3-(3-methyl-
l-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
3-(1,1-difluoroethyl)-N-(4-methyl-3-(3-methyl-l-(6-(methylamino)pyrimidin-4-
yl)-1H-
1,2,4-triazol-5-ylamino)phenyl)benzamide;
N-(4-methyl-3 -(1-(6-(2-(pyrrolidin-l-yl)ethylamino)pyrimidin-4-yl)-1H-1,2,4-
triazol-5-
ylamino)phenyl)-3-(trifluoromethyl)benzamide;
N-(2-tert-butylpyridin-4-yl)-4-methyl-3-(1-(6-(2-(pyrrolidin-l-
yl)ethylamino)pyrimidin-
4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
N-(3-(1-(6-(2-(dimethylamino)ethylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)-
4-methylphenyl)-3-(trifluoromethyl)benzamide;
N-(2-tert-butylpyridin-4-yl)-3-(1-(6-(2-(dimethylamino)ethylamino)pyrimidin-4-
yl)-1H-
1,2,4-triazol-5-ylamino)-4-methylbenzamide;
4-methyl-3-(1-(6-(3-(4-methylpiperazin-1-yl)propylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)-N-(3 -(trifluoromethyl)phenyl)benzamide;
N-(2-tert-butylpyridin-4-yl)-4-methyl-3-(1-(6-(3-(4-methylpiperazin-l-
yl)propylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
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4-methyl-3-(1-(6-(3-morpholinopropylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)-N-(3 -(trifluoromethyl)phenyl)benzamide;
N-(2-tert-butylpyridin-4-yl)-4-methyl-3-(1-(6-(3-
morpholinopropylamino)pyrimidin-4-
yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
4-methyl-3-(1-(6-(1-methylpiperidin-4-ylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-
5-
ylamino)-N-(3 -(trifluoromethyl)phenyl)benzamide;
3-(1-(6-(cyclopropylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-4-methyl-
N-(3-
(trifluoromethyl)phenyl)benzamide;
N-(2-tert-butylpyridin-4-yl)-3-(1-(6-(cyclopropylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)-4-methylbenzamide;
3-(1-(6-(2-methoxyethylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-4-
methyl-N-
(3-(trifluoromethyl)phenyl)benzamide;
N-(2-tert-butylpyridin-4-yl)-3-(1-(6-(2-methoxyethylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)-4-methylbenzamide;
N-(2-tert-butylpyridin-4-yl)-4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)benzamide;
4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-N-(4-
(trifluoromethyl)pyridin-2-yl)benzamide;
N-(4-methyl-3 -(1-(6-(4-methylpiperazin-l-ylamino)pyrimidin-4-yl)-1H-1,2,4-
triazol-5-
ylamino)phenyl)-3-(trifluoromethyl)benzamide;
N-(2-tert-butylpyridin-4-yl)-4-methyl-3-(1-(6-(4-methylpiperazin-l-
ylamino)pyrimidin-
4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
N-(4-methyl-3 -(1-(6-(2-morpholinoethylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-
5-
ylamino)phenyl)-3-(trifluoromethyl)benzamide;
N-(2-tert-butylpyridin-4-yl)-4-methyl-3-(1-(6-(2-
morpholinoethylamino)pyrimidin-4-yl)-
1H-1,2,4-triazol-5-ylamino)benzamide;
4-methyl-3-(1-(6-(4-methylpiperazin-l-ylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-
5-
ylamino)-N-(4-(trifluoromethyl)pyridin-2-yl)benzamide;
4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)-N-(4-(trifluoromethyl)pyridin-2-yl)benzamide;
4-methyl-3-(1-(6-(4-methylpiperazin-1-ylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-
5-
ylamino)-N-(3 -(trifluoromethyl)phenyl)benzamide;
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4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)-N-(3 -(trifluoromethyl)phenyl)benzamide;
2-fluoro-N-(4-methyl-3-(1-(6-(4-methylpiperazin-l-ylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)-5 -(trifluoromethyl)benzamide;
3-fluoro-N-(4-methyl-3-(1-(6-(4-methylpiperazin-l-ylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)-5 -(trifluoromethyl)benzamide;
4-fluoro-N-(4-methyl-3-(1-(6-(4-methylpiperazin-l-ylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)-3 -(trifluoromethyl)benzamide;
4-chloro-N-(4-methyl-3-(1-(6-(4-methylpiperazin-1-ylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)-3 -(trifluoromethyl)benzamide;
2-tert-butyl-N-(4-methyl-3-(1-(6-(4-methylpiperazin-1-ylamino)pyrimidin-4-yl)-
1H-
1,2,4-triazol-5-ylamino)phenyl)isonicotinamide;
2-(1,1-difluoroethyl)-N-(4-methyl-3-(1-(6-(4-methylpiperazin-1-
ylamino)pyrimidin-4-
yl)-1H-1,2,4-triazol-5-ylamino)phenyl)isonicotinamide;
3-(2-cyanopropan-2-yl)-N-(4-methyl-3-(1-(6-(4-methylpiperazin-l-
ylamino)pyrimidin-4-
yl)-1H-1,2,4-triazol-5-ylamino)phenyl)benzamide;
3-(2-methoxypropan-2-yl)-N-(4-methyl-3-(1-(6-(4-methylpiperazin-l-
yl amino)pyrimidin-4-yl) -1 H-1, 2, 4-triazol-5 -ylamino)phenyl)benzamide;
2-fluoro-N-(4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-4-yl)-1H-1,2,4-
triazol-5-ylamino)phenyl)-5 -(trifluoromethyl)benzamide;
3-fluoro-N-(4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-4-yl)-1H-1,2,4-
triazol-5-ylamino)phenyl)-5 -(trifluoromethyl)benzamide;
4-fluoro-N-(4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-4-yl)-1H-1,2,4-
triazol-5-ylamino)phenyl)-3 -(trifluoromethyl)benzamide;
4-chloro-N-(4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-4-yl)-1H-1,2,4-
triazol-5 -ylamino)phenyl)-3 -(trifluoromethyl)benzamide;
2-tert-butyl-N-(4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)isonicotinamide;
2-(1,1-difluoroethyl)-N-(4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-4-
yl)-
1H-1,2,4-triazol-5-ylamino)phenyl)isonicotinamide;
3-(2-cyanopropan-2-yl)-N-(4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-4-
yl)-
1 H-1,2,4-triazol-5-ylamino)phenyl)benzamide;
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3-(2-methoxypropan-2-yl)-N-(4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-
4-
yl)-1H-1,2,4-triazol-5-ylamino)phenyl)benzamide;
3-(4-ethylpiperazin-l-yl)-N-(4-methyl-3-(1-(6-(2-
morpholinoethylamino)pyrimidin-4-
yl)-1H-1,2,4-triazol-5-ylamino)phenyl)-5-(trifluoromethyl)benzamide;
tert-butyl4-(3-(4-methyl-3-(1-(6-(2-morpholinoethylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenylcarbamoyl)-5-(trifluoromethyl)phenyl)piperazine-l-
carboxylate;
N-(4-methyl-3 -(1-(6-(morpholinoamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-3-(trifluoromethyl)benzamide;
4-methyl-3-(1-(6-(morpholinoamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-
N-(3-
(trifluoromethyl)phenyl)benzamide;
N-(4-methyl-3 -(1-(6-(2-(4-methylpiperazin-l-yl)ethylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)phenyl)-3 -(trifluoromethyl)benzamide;
4-methyl-3-(1-(6-(2-(4-methylpiperazin-l-yl)ethylamino)pyrimidin-4-yl)-1H-
1,2,4-
triazol-5-ylamino)-N-(3 -(trifluoromethyl)phenyl)benzamide;
N-(4-methyl-3 -(1-(6-(2-morpholinoethylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-
5-
ylamino)phenyl)-3-(piperazin-l-yl)-5 -(trifluoromethyl)benzamide;
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-methyl-3-(1-(6-(4-methylpiperazin-
l-
ylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
1-(4-fluoro-3-(trifluoromethyl)phenyl)-3-(4-methyl-3-(1-(6-(4-methylpiperazin-
l-
ylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-(1-(6-(2-
morpholinoethylamino)pyrimidin-4-yl)-1 H-1,2,4-triazol-5-ylamino)benzamide;
N-(4-fluoro-3-(trifluoromethyl)phenyl)-4-methyl-3-(1-(6-(2-
morpholinoethylamino)pyrimidin-4-yl)-1 H-1,2,4-triazol-5-ylamino)benzamide;
N-(4-methyl-3 -(3-(methylamino)-1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-
triazol-5-
ylamino)phenyl)-3-(trifluoromethyl)benzamide;
1-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-
3 -(3-(trifluoromethyl)phenyl)urea;
1-(3-fluoro-5-(trifluoromethyl)phenyl)-3-(4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-
yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
1-(4-fluoro-3-(trifluoromethyl)phenyl)-3-(4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-
yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
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1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-
yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
1-(3-(4-ethylpiperazin-l-yl)-5-(trifluoromethyl)phenyl)-3-(4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
1-(4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)phenyl)-
3-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)urea;
N-(3-(2-(dimethylamino)ethoxy)-5-(trifluoromethyl)phenyl)-4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
N-(3-(3-(dimethylamino)propoxy)-5-(trifluoromethyl)phenyl)-4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-N-(3-
(2-(2-
oxopyrrolidin-1-yl)ethoxy)-5 -(trifluoromethyl)phenyl)benzamide;
4-methyl-3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)-N-(3-
(2-
(pyrrolidin-1-yl)ethoxy)-5-(trifluoromethyl)phenyl)benzamide;
N-(3-(2-(diethylamino)ethoxy)-5-(trifluoromethyl)phenyl)-4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide;
1-(2-fluoro-5-(trifluoromethyl)phenyl)-3-(4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-
yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
1-(2-chloro-5-(trifluoromethyl)phenyl)-3-(4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-
yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
1-(3-(4-ethylpiperazin-l-yl)-5-(trifluoromethyl)phenyl)-3-(4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
1-(3-((2-(dimethylamino)ethyl)(methyl)amino)-5-(trifluoromethyl)phenyl)-3-(4-
methyl-
3-(1-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
1-(3-((2-methoxyethyl)(methyl)amino)-5-(trifluoromethyl)phenyl)-3-(4-methyl-3-
(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
1-(3-(2-(dimethylamino)ethoxy)-5-(trifluoromethyl)phenyl)-3-(4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
1-(3-((4-ethylpiperazin-1-yl)methyl)-5-(trifluoromethyl)phenyl)-3-(4-methyl-3-
(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)phenyl)urea;
N-(3-((dimethylamino)methyl)-5-(trifluoromethyl)phenyl)-4-methyl-3-(1-(6-
(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-ylamino)benzamide; or
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3-((3-hydroxyazetidin-1-yl)methyl)-N-(4-methyl-3-(1-(6-(methylamino)pyrimidin-
4-yl)-
IH-1,2,4-triazol-5-ylamino)phenyl)-5-(trifluoromethyl)benzamide, or
pharmaceutically acceptable salts thereof.
[0030] Compounds having Formula (1), (2) or (3) may be useful as protein
kinase inhibitors.
For example, compounds having Formula (1), (2) or (3), and pharmaceutically
acceptable salts,
solvates, N-oxides, prodrugs and isomers thereof, may be used for the
treatment of a kinase-
mediated condition or disease, such as diseases mediated by Alk, Abl, Aurora-
A, B-Raf, C-Raf,
Bcr-Abl, BRK, Blk, Bmx, BTK, C-Kit, C-Src, EphBl, EphB2, EphB4, FLT1, Fms,
F1t3, Fyn,
JAK2, KDR, Lck, Lyn, PDGFRa, PDGFR(3, PKCa, p38 (p38 MAP kinase, SAPK2a), Src,
SIK,
Syk, Tie2 and TrkB kinases, or a combination thereof.
[0031] The compounds of the invention may also be used in combination with a
second
therapeutic agent, for ameliorating a condition mediated by a protein kinase,
such as a B-Raf-
mediated condition. For example, the compounds of the invention may be used in
combination
with a chemotherapeutic agent to treat a cell proliferative disorder,
including but not limited to,
lymphoma, osteosarcoma, melanoma, or breast, renal, prostate, colorectal
(colon), thyroid,
ovarian, pancreatic, neuronal, lung, uterine, gastrointestinal tumor or
chloangiocarcinoma. In
particular embodiments, the compounds of the invention may be used to treat
melanoma, thyroid
cancer, colon cancer, chloangiocarcinoma or ovarian cancer. (See e.g., Davies
et al., Nature
417:949-54 (2002); Brose et al., Cancer Res. 62:6997-7000 (2002); Tuveson et
al., Cancer Cell
4:95-8 (2003); Karasides et al., Oncogene 23:6292-8 (2004)).
[0032] Examples of chemotherapeutic agents which may be used in the
compositions and
methods of the invention include but are not limited to anthracyclines,
alkylating agents (e.g.,
mitomycin C), alkyl sulfonates, aziridines, ethylenimines, methylmelamines,
nitrogen mustards,
nitrosoureas, antibiotics, antimetabolites, folic acid analogs (e.g.,
dihydrofolate reductase
inhibitors such as methotrexate), purine analogs, pyrimidine analogs, enzymes,
podophyllotoxins,
platinum-containing agents, interferons, and interleukins. Particular examples
of known
chemotherapeutic agents which may be used in the compositions and methods of
the invention
include, but are not limited to, busulfan, improsulfan, piposulfan, benzodepa,
carboquone,
meturedepa, uredepa, altretamine, triethylenemelamine,
triethylenephosphoramide,
triethylenethiophosphoramide, trimethylolomelamine, chlorambucil,
chlomaphazine,
cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine
oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil
mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine,
ranimustine, dacarbazine,
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mannomustine, mitobronitol, mitolactol, pipobroman, aclacinomycins,
actinomycin F(1),
anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carzinophilin,
chromomycin,
dactinomycin, daunorubicin, daunomycin, 6-diazo-5-oxo-l-norleucine,
doxorubicin, epirubicin,
mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin,
plicamycin,
porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin, zorubicin,
denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-
mercaptopurine, thiamiprine,
thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,
dideoxyuridine,
doxifluridine, enocitabine, floxuridine, fluorouracil, tegafur, L-
asparaginase, pulmozyme,
aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine,
bestrabucil, bisantrene,
carboplatin, cisplatin, defofamide, demecolcine, diaziquone, elfornithine,
elliptinium acetate,
etoglucid, etoposide, flutamide, gallium nitrate, hydroxyurea, interferon-
alpha, interferon-beta,
interferon-gamma, interleukin-2, lentinan, lonidamine, mitoguazone,
mitoxantrone, mopidamol,
nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-
ethylhydrazide, procarbazine,
razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, teniposide,
tenuazonic acid,
triaziquone, 2,2',2"-trichlorotriethylamine, urethane, vinblastine,
vincristine, and vindesine.
Pharmacology and Utility
[0033] Compounds of the invention are screened against the kinase panel (wild
type and/or
mutation thereof) and may modulate the activity of at least one kinase panel
member. As such,
compounds of the invention may be useful for treating diseases or disorders in
which kinases
contribute to the pathology and/or symptomology of the disease. Examples of
kinases that may
be inhibited by the compounds and compositions described herein and against
which the methods
described herein may be useful include, but are not limited to Alk, Abl,
Aurora-A, B-Raf, C-Raf,
Bcr-Abl, BRK, Blk, Bmx, BTK, C-Kit, C-Src, EphBl, EphB2, EphB4, FLT1, Fms,
F1t3, Fyn,
FRK3, JAK2, KDR, Lck, Lyn, PDGFRa, PDGFR(3, PKCa, p38 (p38 MAP kinase,
SAPK2a),
Src, SIK, Syk, Tie2 and TrkB kinases, and mutant forms thereof.
[0034] The Ras-Raf-MEK-ERK signaling pathway mediates cellular response to
growth
signals. Ras is mutated to an oncogenic form in approximately 15% of human
cancer. The Raf
family belongs to the serine/threonine protein kinase and it includes three
members, A-Raf, B-Raf
and C-Raf (or Raf- 1). The focus on Raf being a drug target has centered on
the relationship of
Raf as a downstream effector of Ras. However, B-Raf may have a prominent role
in the
formation of certain tumors with no requirement for an activated Ras allele
(Nature 417:949-954
(2002). In particular, B-Raf mutations have been detected in a large
percentage of malignant
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melanomas. Existing medical treatments for melanoma are limited in their
effectiveness,
especially for late stage melanomas. The compounds of the present invention
also inhibit cellular
processes involving B-Raf kinase, providing a new therapeutic opportunity for
treatment of
human cancers, such as melanoma.
[0035] Certain abnormal proliferative conditions are believed to be associated
with Raf
expression and are, therefore, believed to be responsive to inhibition of Raf
expression.
Abnormally high levels of expression of the Raf protein are also implicated in
abnormal cell
proliferation. These abnormal proliferative conditions are also believed to be
responsive to
inhibition of Raf kinase expression. For example, expression of the C-Raf
protein is believed to
play a role in abnormal cell proliferation since it has been reported that 60%
of all lung carcinoma
cell lines express unusually high levels of C-Raf mRNA and protein. Further
examples of
abnormal proliferative conditions are hyper-proliferative disorders such as
cancers, hyperplasia,
pulmonary fibrosis, angiogenesis, psoriasis, atherosclerosis and smooth muscle
cell proliferation
in the blood vessels, such as stenosis or restenosis following angioplasty.
The cellular signaling
pathway of which Raf is a part has also been implicated in inflammatory
disorders characterized
by T-cell proliferation (T-cell activation and growth), such as tissue graft
rejection, endotoxin
shock, and glomerular nephritis, for example.
[0036] The compounds of the present invention may also inhibit cellular
processes involving
C-Raf kinase. C-Raf is activated by the Ras oncogene, which is mutated in a
wide number of
human cancers. Therefore inhibition of the kinase activity of C-Raf may
provide a way to
prevent Ras mediated tumor growth [Campbell, S. L., Oncogene, 17, 1395
(1998)].
[0037] The Src family of kinases is implicated in cancer, immune system
dysfunction and
bone remodeling diseases. Members of the Src family include the following
eight kinases in
mammals: Src, Fyn, Yes, Fgr, Lyn, Hck, Lck, and Blk. For general reviews, see
Thomas and
Brugge, Annu. Rev. Cell Dev. Biol. (1997) 13, 513; Lawrence and Niu,
Pharmacol. Ther. (1998)
77, 81; Tatosyan and Mizenina, Biochemistry (Moscow) (2000) 65, 49; Boschelli
et al., Drugs of
the Future 2000, 25(7), 717.
[0038] Fyn encodes a membrane-associated tyrosine kinase that has been
implicated in the
control of cell growth.
[0039] Lck plays a role in T-cell signaling. Mice that lack the Lck gene have
a poor ability to
develop thymocytes. The function of Lck as a positive activator of T-cell
signaling suggests that
Lck inhibitors may be useful for treating autoimmune disease such as
rheumatoid arthritis.
Molina et al., Nature, 357, 161 (1992). Hck, Fgr and Lyn have been identified
as important
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mediators of integrin signaling in myeloid leukocytes. Lowell et al., J.
Leukoc. Diol., 65, 313
(1999). Inhibition of these kinase mediators may therefore be useful for
treating inflammation.
Boschelli et al., Drugs of the Future, 2000, 25(7), 717.
[0040] Lyn, a member of the Src family, plays a role in the regulation of B-
cell immune
responses. Lyn-deficient mice display disrupted B-cell function, leading to
autoimmunity and
defective mast cell degranulation. Studies have also suggested that Lyn is a
negative regulator of
apoptosis in various cell systems. In leukemic cells, Lyn is constitutively
activated, and the
inhibition of Lyn expression reversed proliferation. In addition, Lyn has been
shown to be
expressed in colon and PC cells, and that overexpression of a dominant active
Lyn in colon cancer
cell lines induced chemoresistance. (Goldenberg-Furmanov et al., Cancer Res.
64:1058-1066
(2004)).
[0041] The kinase, c-Src transmits oncogenic signals of many receptors. For
example, over-
expression of EGFR or HER2/neu in tumors leads to the constitutive activation
of C-Src, which is
characteristic for the malignant cell but absent from the normal cell. On the
other hand, mice
deficient in the expression of C-Src exhibit an osteopetrotic phenotype,
indicating a key
participation of C-Src in osteoclast function and a possible involvement in
related disorders. C-
Src tyrosine kinase (CSK) influences the metastatic potential of cancer cells,
particularly colon
cancer.
[0042] C-Kit has a substantial homology to the PDGF receptor and to the CSF-1
receptor (c-
Fms). Investigations on various erythroid and myeloid cell lines indicate an
expression of the C-
Kit gene in early stages of differentiation (Andre et al., Oncogene 4 (1989),
1047-1049). Certain
tumors such as glioblastoma cells likewise exhibit a pronounced expression of
the C-Kit gene.
[0043] Eph receptors, which include EphA and EphB subfamily, consist of the
largest group
of receptor tyrosine kinases. EphB was found to be overexpressed in several
tumors including
ovarian tumors, liver tumors, kidney tumors as well as melanomas.
Downregulation of EphB
signaling has shown to inhibit tumor growth and metastasis. Therefore, EphB
may be an
important target for anti-tumorigenic therapies. (Clevers et al., Cancer Res.
66:2-5 (2006);
Heroult et al., Experimental Cell Res. 312: 642-650 (2006); and Batlle et al.,
Nature 435:1126-
1130 (2005)).
[0044] Kinase insert domain-containing receptor (referred to as "KDR"
hereinafter) [WO
92/14748; Proc. Natl. Acad. Sci. USA, 88: 9026 (1991)]; Biochem. Biophys. Res.
Comm., 187:
1579 (1992); WO 94/11499) and Fms-like tyrosine kinase (referred to as "Fltl"
hereinafter)
[Oncogene, 5: 519 (1990); Science, 255: 989 (1992)] belong to the receptor
type tyrosine kinase
18
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family. It has been reported that VEGF specifically binds to Flt-1 and KDR at
Kd values of 20
pM and 75 pM and that Fltl and KDR are expressed in vascular endothelial cells
in a specific
manner [Proc. Natl. Acad. Sci. USA, 90: 7533 (1993); Proc. Natl. Acad. Sci.
USA, 90: 8915
(1993)]. With regard to Flt-1 in various diseases, it has been reported that,
in comparison with
vascular endothelial cells in normal tissues, expression of Flt-1 mRNA
increases in tumor
vascular endothelial cells of human glioblastoma tissues [Nature, 359: 845
(1992)] and tumor
vascular endothelial cells of human digestive organ cancer tissues [Cancer
Research, 53: 4727
(1993)]. Additionally, it has been reported that expression of Flt-1 mRNA is
observed by in situ
hybridization in vascular endothelial cells of joints of patients with
rheumatoid arthritis [J.
Experimental Medicine, 180: 341 (1994)]. Studies also suggest that Flt-1 plays
an important role
in tumor angiogenesis.
[0045] F1t3 is a member of the type III receptor tyrosine kinase (RTK) family.
F1t3 (Fms-like
tyrosine kinase) is also known as Flk-2 (fetal liver kinase 2). Aberrant
expression of the F1t3
gene has been documented in both adult and childhood leukemias including acute
myeloid
leukemia (AML), AML with trilineage myelodysplasia (AML/TMDS), acute
lymphoblastic
leukemia (ALL), and myelodysplastic syndrome (MDS). In approximately 25% of
AML, the
leukemia cells express a constitutively active form of auto-phosphorylated (p)
FLT3 tyrosine
kinase on the cell surface. The activity of p-FLT3 confers growth and survival
advantage on the
leukemic cells. Inhibition of p-FLT3 kinase activity induces apoptosis
(programmed cell death)
of the leukemic cells.
[0046] Abelson tyrosine kinase (i.e. Abl, c-Abl) is involved in the regulation
of the cell cycle,
in the cellular response to genotoxic stress, and in the transmission of
information about the
cellular environment through integrin signaling. The Abl protein appears to
serve a complex role
as a cellular module that integrates signals from various extracellular and
intracellular sources
and that influences decisions in regard to cell cycle and apoptosis. Abelson
tyrosine kinase
includes sub-types derivatives such as the chimeric fusion (oncoprotein) Bcr-
Abl with
deregulated tyrosine kinase activity or the v-Abl. Bcr-Abl is important in the
pathogenesis of
95% of chronic myelogenous leukemia (CML) and 10% of acute lymphocytic
leukemia.
[0047] Compounds of the present invention may inhibit Abl kinase, for example,
v-Abl
kinase. The compounds of the present invention may also inhibit wild-type Bcr-
Abl kinase and
mutations of Bcr-Abl kinase, and thus may be suitable for the treatment of Bcr-
Abl-positive
cancer and tumor diseases, such as leukemias (e.g., chronic myeloid leukemia
and acute
lymphoblastic leukemia) and other proliferation disorders related to Bcr-Abl.
Compounds of the
19
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present invention may also be effective against leukemic stem cells, and may
be potentially useful
for the purification of these cells in vitro after removal of said cells (for
example, bone marrow
removal), and reimplantation of the cells once they have been cleared of
cancer cells (for
example, reimplantation of purified bone marrow cells).
[0048] Anaplastic lymphoma kinase (ALK), a member of the insulin receptor
superfamily of
receptor tyrosine kinases, has been implicated in oncogenesis in hematopoietic
and non-
hematopoietic tumors. The aberrant expression of full-length ALK receptor
proteins has been
reported in neuroblastomas and glioblastomas; and ALK fusion proteins have
occurred in
anaplastic large cell lymphoma. The study of ALK fusion proteins has also
raised the possibility
of new therapeutic treatments for patients with ALK-positive malignancies.
(Pulford et al., Cell.
Mol. Life Sci. 61:2939-2953 (2004)).
[0049] Aurora-A, a serine/threonine mitotic kinase, has been reported to be
overexpressed in
various human cancers, and its overexpression induces aneuploidy, centrosome
amplification and
tumorigenic transformation in cultured human and rodent cells. (Zhang et al.,
Oncogene
23:8720-30 (2004)).
[0050] Bmx/Etk non-receptor tyrosine protein kinase has been implicated in
endothelial cell
migration and tube formation in vitro. Bmx in endothelium and bone marrow has
also been
reported to play an important role in arteriogenesis and angiogenesis in vivo,
suggesting that Bmx
may be a novel target for the treatment of vascular diseases such as coronary
artery disease and
peripheral arterial disease. (He et al., J. Clin. Invest. 116:2344-2355
(2006)).
[0051] Bruton's tyrosine kinase (BTK) gene encodes a cytoplasmic tyrosine
kinase that plays
an essential role in mediating BCR signaling. (de Weers et al., J. Biol. Chem.
269:23857-23860
(1994); Kurosaki et al., Immunity. 12:1-5 (2000)). Defects in the BTK gene
cause
Agammaglobulinemia, an X-linked immunodeficiency characterized by failure to
produce mature
B lymphocyte cells and associated with a failure of Ig heavy chain
rearrangement.
[0052] Breast tumor kinase (Brk) is a soluble protein-tyrosine kinase
overexpressed in the
majority of breast cancers and also in normal skin and gut epithelium, but not
in normal breast
epithelial cells. (Zhang et al., J Biol. Chem. 280:1982-1991 (2005)).
[0053] The Janus kinases (JAK) are a family of tyrosine kinases consisting of
JAK1, JAK2,
JAK3 and TYK2. The JAKs play an important role in cytokine signaling. The down-
stream
substrates of the JAK family of kinases include the signal transducer and
activator of transcription
(STAT) proteins. JAK/STAT signaling has been implicated in the mediation of
many abnormal
immune responses such as allergies, asthma, autoimmune diseases such as
transplant rejection,
CA 02690653 2009-12-14
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rheumatoid arthritis, amyotrophic lateral sclerosis and multiple sclerosis, as
well as in solid and
hematologic malignancies such as leukemias and lymphomas.
[0054] An important factor in the tumor angiogenesis is vascular endothelium
growth
factor(VEGF). VEGF can promote and maintain the establishment of tumor
vascular system, and
can also promote the tumor growth directly. VEGF can induce the mitogenesis
and chemotaxis
of vascular endothelial cell(VEC) and tumor cell (TC). Almost all types of TC
can secret VEGF,
but the expression of VEGF in the normal tissue is very low. In the four VEGF
receptors, KDR is
the main receptor which gives play to VEGF functions. KDR is highly expressed
on tumor VEC
while lowly expressed on the normal tissues. (Ren et al., World J.
Gastroentrol. 8:596-601
(2002)).
[0055] Mitogen-activated protein kinases (MAPKs) are members of conserved
signal
transduction pathways that activate transcription factors, translation factors
and other target
molecules in response to a variety of extracellular signals. MAPKs are
activated by
phosphorylation at a dual phosphorylation motif having the sequence Thr-X-Tyr
by mitogen-
activated protein kinase kinases (MKKs). In higher eukaryotes, the
physiological role of MAPK
signaling has been correlated with cellular events such as proliferation,
oncogenesis, development
and differentiation. Accordingly, the ability to regulate signal transduction
via these pathways
(particularly via MKK4 and MKK6) could lead to the development of treatments
and preventive
therapies for human diseases associated with MAPK signaling, such as
inflammatory diseases,
autoimmune diseases and cancer.
[0056] Multiple forms of p38 MAPK (a, (3, y, S), each encoded by a separate
gene, form part
of a kinase cascade involved in the response of cells to a variety of stimuli,
including osmotic
stress, UV light and cytokine mediated events. These four isoforms of p38 are
thought to regulate
different aspects of intracellular signaling. Its activation is part of a
cascade of signaling events
that lead to the synthesis and production of pro-inflammatory cytokines like
TNFa. P38
functions by phosphorylating downstream substrates that include other kinases
and transcription
factors. Agents that inhibit p38 kinase have been shown to block the
production of cytokines,
including but not limited to TNFa, IL-6, IL-8 and IL-10. Peripheral blood
monocytes (PBMCs)
have been shown to express and secrete pro-inflammatory cytokines when
stimulated with
lipopolysaccharide (LPS) in vitro. P38 inhibitors efficiently block this
effect when PBMCs are
pretreated with such compounds prior to stimulation with LPS. P38 inhibitors
are efficacious in
animal models of inflammatory disease. The destructive effects of many disease
states are caused
21
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by the over production of pro-inflammatory cytokines. The ability of p38
inhibitors to regulate
this overproduction makes them useful as disease modifying agents.
[0057] Molecules that block p38's function have been shown to be effective in
inhibiting bone
resorption, inflammation, and other immune and inflammation-based pathologies.
Therefore,
compounds of the invention that inhibit p38 activity are useful for the
treatment of inflammation,
osteoarthritis, rheumatoid arthritis, cancer, autoimmune diseases, and for the
treatment of other
cytokine mediated diseases.
[0058] PDGF (Platelet-derived Growth Factor) is a commonly occurring growth
factor, which
plays an important role both in normal cell growth and pathological cell
proliferation, as seen in
carcinogenesis and in diseases of the smooth-muscle cells of blood vessels,
for example in
atherosclerosis and thrombosis. Compounds of the invention may inhibit PDGF
receptor
(PDGFR) activity, and may therefore be suitable for the treatment of tumor
diseases, such as
gliomas, sarcomas, prostate tumors, and tumors of the colon, breast, and
ovary.
[0059] Compounds of the present invention, may be used not only for treating
tumors, for
example in small cell lung cancer, but also as an agent to treat non-malignant
proliferative
disorders, such as atherosclerosis, thrombosis, psoriasis, scleroderma and
fibrosis. Compounds of
the present invention may also be useful for the protection of stem cells, for
example to combat
the hemotoxic effect of chemotherapeutic agents, such as 5-fluoruracil, and in
asthma.
Compounds of the invention may especially be used for the treatment of
diseases due to the
overexpression of PDGF receptor kinase.
[0060] Compounds of the present invention may exhibit useful effects in the
treatment of
disorders arising as a result of transplantation, for example, allogenic
transplantation, especially
tissue rejection, such as obliterative bronchiolitis (OB), i.e. a chronic
rejection of allogenic lung
transplants. In contrast to patients without OB, those with OB often show an
elevated PDGF
level in bronchoalveolar lavage fluids.
[0061] Compounds of the present invention may also be effective against
diseases associated
with vascular smooth-muscle cell migration and proliferation (where PDGF and
PDGFR often
also play a role), such as restenosis and atherosclerosis. These effects and
the consequences
thereof for the proliferation or migration of vascular smooth-muscle cells in
vitro and in vivo may
be demonstrated by administration of the compounds of the present invention,
and also by
investigating its effect on the thickening of the vascular intima following
mechanical injury in
vivo.
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[0062] Protein kinase C (PKC) functions in processes relevant to
carcinogenesis, tumor cell
metastasis, and apoptosis. PKCa is associated with a diverse range of cancers,
and is previously
shown to be overexpressed in three out of four antiestrogen resistant breast
cancer cell lines.
(Frankel et al., Breast Cancer Res Treat. 2006 Oct. 24 (ePub)).
[0063] The stress activated protein kinases (SAPKs) are a family of protein
kinases that
represent the penultimate step in signal transduction pathways that result in
activation of the c-Jun
transcription factor and expression of genes regulated by c-Jun. In
particular, c-Jun is involved in
the transcription of genes that encode proteins involved in the repair of DNA
that is damaged due
to genotoxic insults. Therefore, agents that inhibit SAPK activity in a cell
prevent DNA repair
and sensitize the cell to agents that induce DNA damage or inhibit DNA
synthesis and induce
apoptosis of a cell or that inhibit cell proliferation.
[0064] The region encompassing the SNFILK locus (also known as SIK) has been
implicated
in congenital heart defects often observed in patients with Down syndrome.
Snfllk is also
expressed in skeletal muscle progenitor cells of the somite beginning at 9.5
dpc, suggesting a
more general role for SNFILK in the earliest stages of muscle growth and/or
differentiation.
(Genomics 83:1105-15 (2004)).
[0065] Syk is a tyrosine kinase that plays an important role in mast cell
degranulation and
eosinophil activation. Accordingly, Syk kinase is implicated in various
allergic disorders, in
particular asthma. It has been shown that Syk binds to the phosphorylated
gamma chain of the
FccRl receptor via N-terminal SH2 domains, and is important for downstream
signaling.
[0066] An inhibition of tumor growth and vascularization, and a decrease in
lung metastases
during adenoviral infections or during injections of the extracellular domain
of Tie-2 (Tek)have
been shown in breast tumor and melanoma xenograft models. (Lin et al., J.
Clin. Invest. 100, 8:
2072-2078 (1997) and P. Lin, PNAS 95, 8829-8834, (1998)). Tie2 inhibitors can
be used in
situations where neovascularization takes place inappropriately (i.e. in
diabetic retinopathy,
chronic inflammation, psoriasis, Kaposi's sarcoma, chronic neovascularization
due to macular
degeneration, rheumatoid arthritis, infantile haemangioma and cancers).
[0067] The Trk family of neurotrophin receptors (TrkA, TrkB, TrkC) promotes
the survival,
growth and differentiation of the neuronal and non-neuronal tissues. The TrkB
protein is
expressed in neuroendocrine-type cells in the small intestine and colon, in
the alpha cells of the
pancreas, in the monocytes and macrophages of the lymph nodes and of the
spleen, and in the
granular layers of the epidermis . Expression of the TrkB protein has been
associated with an
unfavorable progression of Wilms tumors and of neuroblastomas. Moreover, TrkB
is expressed
23
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in cancerous prostate cells but not in normal cells. The signaling pathway
downstream of the Trk
receptors involves the cascade of MAPK activation through the Shc, activated
Ras, ERK-1 and
ERK-2 genes, and the PLC-gamma transduction pathway (Sugimoto et al., Jpn J.
Cancer Res.
2001 Feb; 92(2):152-60).
[0068] The class III receptor tyrosine kinases (RTKs), which include C-Fms, C-
Kit, FLT3,
platelet-derived growth factor receptor a(PDGFR(x) and APDGFR)6), have been
reported to be
associated with the pathogenesis of an increasing number of malignancies.
(Blume-Jensen et al.,
Nature 411:355-565 (2001); Scheijin et al., Oncogene 21:3314-3333 (2002)).
[0069] In accordance with the foregoing, the present invention further
provides a method for
preventing or treating any of the diseases or disorders described above in a
subject in need of
such treatment, which method comprises administering to said subject a
therapeutically effective
amount of a compound of Formula (1), (2) or (3), or a pharmaceutically
acceptable salt thereof.
For any of the above uses, the required dosage will vary depending on the mode
of
administration, the particular condition to be treated and the effect desired.
(See, "Administration
and Pharmaceutical Compositions," infra)
Administration and Pharmaceutical Compositions
[0070] In general, compounds of the invention will be administered in
therapeutically
effective amounts via any of the usual and acceptable modes known in the art,
either singly or in
combination with one or more therapeutic agents. A therapeutically effective
amount may vary
widely depending on the severity of the disease, the age and relative health
of the subject, the
potency of the compound used and other factors. In general, satisfactory
results are indicated to
be obtained systemically at daily dosages of from about 0.03 to 2.5 mg/kg per
body weight. An
indicated daily dosage in the larger mammal, e.g. humans, is in the range from
about 0.5 mg to
about 100 mg, conveniently administered, e.g. in divided doses up to four
times a day or in retard
form. Suitable unit dosage forms for oral administration comprise from ca. 1
to 50 mg active
ingredient.
[0071] Compounds of the invention may be administered as pharmaceutical
compositions by
any conventional route, in particular enterally, e.g., orally in the form of
tablets or capsules;
parenterally, e.g., in the form of injectable solutions or suspensions;
topically, e.g., in the form of
lotions, gels, ointments or creams; or in a nasal or suppository form.
[0072] Pharmaceutical compositions comprising a compound of the present
invention in free
form or in a pharmaceutically acceptable salt form in association with at
least one
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pharmaceutically acceptable carrier or diluent may be manufactured in a
conventional manner by
mixing, granulating or coating methods. For example, oral compositions can be
tablets or gelatin
capsules comprising the active ingredient together with a) diluents, e.g.,
lactose, dextrose,
sucrose, mannitol, sorbitol, cellulose and/or glycine; and/or b) lubricants,
e.g., silica, talcum,
stearic acid or its magnesium or calcium salt and/or polyethylene glycol.
Tablets may further
comprise c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin,
tragacanth,
methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone;
and if desired, d)
disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or
effervescent mixtures; and/or
e) absorbents, colorants, flavors and sweeteners. Injectable compositions can
be aqueous isotonic
solutions or suspensions, and suppositories can be prepared from fatty
emulsions or suspensions.
[0073] The compositions may be sterilized and/or contain adjuvants, such as
preserving,
stabilizing, wetting or emulsifying agents, solution promoters, salts for
regulating the osmotic
pressure and/or buffers. In addition, they may also contain other
therapeutically valuable
substances. Suitable formulations for transdermal applications include an
effective amount of a
compound of the present invention with a carrier. A carrier can include
absorbable
pharmacologically acceptable solvents to assist passage through the skin of
the host. For
example, transdermal devices are in the form of a bandage comprising a backing
member, a
reservoir containing the compound optionally with carriers, optionally a rate
controlling barrier to
deliver the compound to the skin of the host at a controlled and predetermined
rate over a
prolonged period of time, and means to secure the device to the skin. Matrix
transdermal
formulations may also be used. Suitable formulations for topical application,
e.g., to the skin and
eyes, may be aqueous solutions, ointments, creams or gels well-known in the
art. Such may
contain solubilizers, stabilizers, tonicity enhancing agents, buffers and
preservatives.
[0074] Compounds of the invention may be administered in therapeutically
effective amounts
in combination with one or more therapeutic agents (pharmaceutical
combinations). For
example, synergistic effects can occur with other immunomodulatory or anti-
inflammatory
substances, for example when used in combination with cyclosporin, rapamycin,
or ascomycin, or
immunosuppressant analogues thereof, for example cyclosporin A(CsA),
cyclosporin G, FK-506,
rapamycin, or comparable compounds, corticosteroids, cyclophosphamide,
azathioprine,
methotrexate, brequinar, leflunomide, mizoribine, mycophenolic acid,
mycophenolate mofetil,
15-deoxyspergualin, immunosuppressant antibodies, especially monoclonal
antibodies for
leukocyte receptors, for example MHC, CD2, CD3, CD4, CD7, CD25, CD28, B7,
CD45, CD58
or their ligands, or other immunomodulatory compounds, such as CTLA41g. Where
the
CA 02690653 2009-12-14
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compounds of the invention are administered in conjunction with other
therapies, dosages of the
co-administered compounds will of course vary depending on the type of co-drug
employed, on
the specific drug employed, on the condition being treated and so forth.
[0075] The invention also provides for a pharmaceutical combinations, e.g. a
kit, comprising
a) a first agent which is a compound of the invention as disclosed herein, in
free form or in
pharmaceutically acceptable salt form, and b) at least one co-agent. The kit
can comprise
instructions for its administration.
Processes for Making Compounds of the Invention
[0076] General procedures for preparing compounds of the invention are
described in the
Examples, infra. In the reactions described, reactive functional groups, for
example hydroxy,
amino, imino, thio or carboxy groups, where these are desired in the final
product, may be
protected to avoid their unwanted participation in the reactions. Conventional
protecting groups
may be used in accordance with standard practice (see e.g., T.W. Greene and P.
G. M. Wuts in
"Protective Groups in Organic Chemistry", John Wiley and Sons, 1991).
[0077] A compound of the invention may be prepared as a pharmaceutically
acceptable acid
addition salt by reacting the free base form of the compound with a
pharmaceutically acceptable
inorganic or organic acid. Alternatively, a pharmaceutically acceptable base
addition salt of a
compound of the invention may be prepared by reacting the free acid form of
the compound with
a pharmaceutically acceptable inorganic or organic base. Alternatively, the
salt forms of the
compounds of the invention may be prepared using salts of the starting
materials or intermediates.
[0078] The free acid or free base forms of the compounds of the invention may
be prepared
from the corresponding base addition salt or acid addition salt from,
respectively. For example, a
compound of the invention in an acid addition salt form may be converted to
the corresponding
free base by treating with a suitable base (e.g., ammonium hydroxide solution,
sodium hydroxide,
and the like). A compound of the invention in a base addition salt form may be
converted to the
corresponding free acid by treating with a suitable acid (e.g., hydrochloric
acid, etc.).
[0079] Compounds of the invention in unoxidized form may be prepared from N-
oxides of
compounds of the invention by treating with a reducing agent (e.g., sulfur,
sulfur dioxide,
triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus
trichloride,
tribromide, or the like) in a suitable inert organic solvent (e.g.
acetonitrile, ethanol, aqueous
dioxane, or the like) at 0 to 80 C.
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[0080] Prodrug derivatives of the compounds of the invention may be prepared
by methods
known to those of ordinary skill in the art (See e.g., Saulnier et al.,
(1994), Bioorganic and
Medicinal Chemistry Letters, Vol. 4, p. 1985). For example, appropriate
prodrugs may be
prepared by reacting a non-derivatized compound of the invention with a
suitable carbamylating
agent (e.g., 1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or
the like).
[0081] Compounds of the present invention may be conveniently prepared or
formed during
the process of the invention, as solvates (e.g., hydrates). Hydrates of
compounds of the present
invention may be conveniently prepared by recrystallization from an
aqueous/organic solvent
mixture, using organic solvents such as dioxin, tetrahydrofuran or methanol.
[0082] Compounds of the invention may be prepared as their individual
stereoisomers by
reacting a racemic mixture of the compound with an optically active resolving
agent to form a
pair of diastereoisomeric compounds, separating the diastereomers and
recovering the optically
pure enantiomers. Resolution of enantiomers may be carried out using covalent
diastereomeric
derivatives of the compounds of the invention, or by using dissociable
complexes (e.g.,
crystalline diastereomeric salts). Diastereomers have distinct physical
properties (e.g., melting
points, boiling points, solubility, reactivity, etc.), and may be readily
separated by taking
advantage of these dissimilarities. The diastereomers may be separated by
chromatography, or by
separation/resolution techniques based upon differences in solubility. The
optically pure
enantiomer is then recovered, along with the resolving agent, by any practical
means that would
not result in racemization. A more detailed description of the techniques
applicable to the
resolution of stereoisomers of compounds from their racemic mixture can be
found in Jean
Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and
Resolutions", John Wiley
And Sons, Inc., 1981.
[0083] In summary, compounds having Formula (1), (2) or (3) may be made by a
process as
described in the Examples; and
(a) optionally converting a compound of the invention into a pharmaceutically
acceptable salt;
(b) optionally converting a salt form of a compound of the invention to a non-
salt form;
(c) optionally converting an unoxidized form of a compound of the invention
into a
pharmaceutically acceptable N-oxide;
(d) optionally converting an N-oxide form of a compound of the invention to
its
unoxidized form;
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(e) optionally resolving an individual isomer of a compound of the invention
from a
mixture of isomers;
(f) optionally converting a non-derivatized compound of the invention into a
pharmaceutically acceptable prodrug derivative; and
(g) optionally converting a prodrug derivative of a compound of the invention
to its non-
derivatized form.
[0084] The present invention also includes all suitable isotopic variations of
the compounds
of the invention, or pharmaceutically acceptable salts thereof. An isotopic
variation of a
compound of the invention or a pharmaceutically acceptable salt thereof is
defined as one in
which at least one atom is replaced by an atom having the same atomic number
but an atomic
mass different from the atomic mass usually found in nature. Examples of
isotopes that may be
incorporated into the compounds of the invention and pharmaceutically
acceptable salts thereof
include but are not limited to isotopes of hydrogen, carbon, nitrogen and
oxygen such as as 2 H,
3H 11C 13C 14C 15N 170 180,35S, 18F 36C1 and 123I. Certain isotopic variations
of the
compounds of the invention and pharmaceutically acceptable salts thereof, for
example, those in
which a radioactive isotope such as 3H or 14C is incorporated, are useful in
drug and/or substrate
tissue distribution studies. In particular examples, 3H and 14C isotopes may
be used for their ease
of preparation and detectability. In other examples, substitution with
isotopes such as 2 H may
afford certain therapeutic advantages resulting from greater metabolic
stability, such as increased
in vivo half-life or reduced dosage requirements. Isotopic variations of the
compounds of the
invention or pharmaceutically acceptable salts thereof can generally be
prepared by conventional
procedures using appropriate isotopic variations of suitable reagents.
Isotopic variations of the
compounds have the potential to change a compound's metabolic fate and/or
create small changes
in physical properties such as hydrophobicity, and the like. Isotopic
variation have the potential
to enhance efficacy and safety, enhance bioavailability and half-life, alter
protein binding, change
biodistribution, increase the proportion of active metabolites and/or decrease
the formation of
reactive or toxic metabolites.
[0085] Insofar as the production of the starting materials is not particularly
described, the
compounds are known or may be prepared analogously to methods known in the art
or as
disclosed in the Examples hereinafter. One of skill in the art will appreciate
that the above
transformations are only representative of methods for preparation of the
compounds of the
present invention, and that other well known methods can similarly be used.
[0086] The following examples are offered to illustrate but not to limit the
invention.
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Example 1
N-{4-Methyl-3-[2-(6-methylamino-pyrimidin-4-yl)-2H-[1,2,4ltriazol-3-ylaminol-
phen 1~1-3-
trifluoromethyl-benzamide (5)
NN NH2 NN NH2 NN NH2
'ki / Cs2CO3 ll ~ CH3NH2 ll
CI' CI + HN \ N ~ CI/ / N\ N MeOH HN/ ~/ N~ N
1 2
/ I
Pd(OAc)2, Cs2CO3 N~N H \ NHBoc TFA:DCM/1:1
4,5-Bis(diphenylphosphine)-9,9-diemethylxanthane HN" v NIN
~ N=l
3
F F
F / I O F F
N~N H':a NH2 O /\ N~N HN \ NH~F
k/~ _ ~ ~ I
HN N~N HO HNk/N~ N /
I N~ HATU, EtN(iso-Pr)2 I N=~
4 5
2-(6-Chloro-pyrimidin-4-yl)-2H-[1,2,4ltriazol-3-ylamine (1)
N^N NH2
CI~'~N/~N
N=~
[0087] To a nitrogen purged flask is added 4,6-dichloropyrimidine (20 g, 0.134
mol), 3-
amino-1,2,4-triazole (11.3 g, 0.134 mol), CszCO3 (43.7 g, 0.134 mol) and DMF
(300 mL). After
stirring at rt for 2 days, the mixture is diluted with EtOAc (800 mL), then
washed with water (3x).
The organic layer is dried with MgSO4 and concentrated in vacuo. The residue
is dissolved in hot
CH3CN (800 mL) and kept at rt overnight. The undesired isomer is precipitated
out and removed
by filtration. The filtrate is concentrated slowly at 70 C until a yellow
solid starts to precipitate
out, then the solution is kept at rt overnight. The precipitate is collected
by filtration, checked by
TLC, and recrystallized as needed. 1H NMR (400 MHz, DMSO) 8 8.90 (s, 1H), 7.90
(s, 2H), 7.80
(s, 1H), 7.79 (s, 1H). MS (ESI) m/z: 197 (M+H)+.
29
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[6-(5-Amino-[1,2,4ltriazol-l-yl) -pyrimidin-4-yll -methyl-amine (2)
N~ N "\2
HN ~ N ~N
[0088] To a high pressure tube is added compound 1 (1.0 g), MeOH (20 mL) and
CH3NH2
(20 mL, 1 M soln in MeOH). The mixture is heated to 50 C for 2 hrs, then
concentrated in vacuo,
and the residue is purified by flash chromatography [silica gel, DCM:MeOH/9:
1]. 1H NMR (400
MHz, DMSO) 8 8.40 (s, 1H), 7.60 (s, 2H), 7.56 (s, 2H), 6.70 (s, 1H), 2.50 (s,
3H). MS (ESI) m/z:
192 (M+H)+.
{4-Methyl-3-[2-(6-methylamino-pyrimidin-4-yl)-2H- [ 1,2,4ltriazol-3-ylaminol-
phenyl} -
carbamic acid tert-butyl ester (3)
~ I
NN HN \ NHBoc
I
HIN N=/
[0089] To a high pressure tube is added compound 2 (0.708 g, 3.71 mmol), (3-
Bromo-4-
methyl-phenyl)-carbamic acid tert-butyl ester (0.909 g, 4.08 mmol), Pd(OAc)2
(83 mg, 0.37
mmol), CszCO3 (1.207 g, 3.70 mmol), 4,5-Bis(diphenylphosphine)-9,9-
dimethylxanthane (0.214
g, 0.37 mmol) and 1,4-dioxane (10 mL). The mixture is flushed with N2 at 0 C
for a few minutes,
and heated to 90 C overnight. The mixture is then poured into H20 and
extracted with EtOAc.
The organic layer is separated, dried with MgS04, then concentrated in vacuo
and the residue is
purified by flash chromatography [silica gel, hexane:EtOAc/4:6]. MS (ESI) m/z:
397 (M+H)+.
4-Methyl-N3- [2-(6-methylamino-pyrimidin-4-yl)-2H- [ 1,2,4ltriazol-3-yll-
benzene-1,3-
diamine (4)
/ I
N N HN \ NH2
H N ~ N~~ N
I N
[0090] Compound 3 (0.80 g) is dissolved in a mixed solvent of DCM:TFA (10
mL:10 mL).
The mixture is stirred at rt for 1 hr, then concentrated in vacuo. The residue
is dissolved in
CA 02690653 2009-12-14
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EtOAc, then washed with aqueous NaHCO3. The organic layer is dried with MgSO4,
then
concentrated to give a yellow solid. MS (ESI) m/z: 297 (M+H)+.
N- { 4-Methyl-3- [2-(6-methylamino-pyrimidin-4-yl)-2H- [ 1,2,4]triazol-3-
ylaminol-
phenyl} -3 -trifluoromethyl-benzamide (5)
aNH O F N^N HN F
HN" v _N" `N
[0091] To a vial is added compound 4 (20 mg, 0.067 mmol), 3-trifluorobenzoic
acid (13 mg,
0.067 mmol), HATU (26 mg, 0.067 mmol), EtN(iPr)2 (11.7 L, 0.122 mmol) and DMF
(2 mL).
The mixture is stirred at rt overnight, then purified by HPLC. 1H NMR (400
MHz, DMSO) 8
11.20 (s, 1H), 10.50 (s, 1H), 8.70 (s, 1H), 8.55 (s, 1H), 8.30 (s, 1H), 8.28
(d, 1H), 7.95 (s, 1H),
7.90 (s, 1H), 7.80 (t, 1H), 7.45 (d, 1H), 7.26 (d, 1H), 6.85 (s, 1H), 2.90 (s,
3H), 2.40 (s, 3H). MS
(ESI) m/z: 469 (M+H)+.
Example 2
4-Methyl-3- [2-(6-methylamino-pyrimidin-4-yl)-2H- [ 1,2,4]triazol-3-ylaminol-N-
(3-
trifluoromethyl-phenyl)-benzamide (8)
~ /
~ NH2 ^ ~ I O
~ ~ Br Pd(OAc)z, CszCO3 N1N HN ~
HN / N ~N ~ /~ ~ O
~ N=J HN ~% `N/\\N
4,5-Bis(diphenylphosphine)-9,9-diemethylxanthane ~ N=l
2
6
F F F F
/ ~ HzN F F
LiOH, THF:H20/1:1 NII~~NI HN \ OH I/ N~N HN \ I NH~\
~%~ '\ O ~ /
H ~ N~ HATU, EtN(iso-Pr)z H i / NI~IN O
N-
7 8
31
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4-Methyl-3- [2-(6-methylamino-pyrimidin-4-yl)-2H- [ 1,2,4]triazol-3-ylaminol-
benzoic
acid methyl ester (6)
N^N HN O~
HN" ~N" `N O
1 N-/
[0092] To a solution of 2 (1.82 g, 9.52mmol) in dioxane (95 mL) is added
Pd2(dba)3 (435 mg,
0.47 mmol), XantPhos (826 mg,1.42 mmol), CsZCO3 (7.44 g, 22.8 mmol) and 3-
bromo-4-methyl-
benzoic acid methyl ester (2.4 g, 10.5 mmol). The flask is purged with Argon,
capped and heated
at 90 C overnight. The mixture is diluted with H20 and extracted with ethyl
acetate. The organic
phase are washed with brine, dried over anhydrous Na2SO4, filtered and
concentrated in vacuo.
Flash column chromatography on silica gel (hexane:EtOAc/1:1) provided compound
6. MS (ESI)
m/z: 340.1 (M + H)+.
4-Methyl-3- [2-(6-methylamino-pyrimidin-4-yl)-2H- [ 1,2,4]triazol-3-ylaminol-
benzoic
acid (7)
NN HN OH
HN" v _N" `N 0
1 N-/
[0093] A mixture of compound 6 (800 mg, 2.36 mg) and LiOH=HZO (990 mg, 2.6
mmol) in
THF:HZO/3:1 (24 mL) is stirred at rt overnight. The reaction mixture is
concentrated in vacuo to
remove most of THF. The mixture is cooled down to 0 C and filtered to give the
desired product
7 as a sodium salt. MS (ESI) m/z: 326.1 (M+H)+.
32
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4-Methyl-3- [2-(6-methylamino-pyrimidin-4-yl)-2H- [ 1,2,4]triazol-3-ylaminol-N-
(3-
trifluoromethyl-phenyl)-benzamide (8)
F F
H F
NN HN N
HN" v _N N O
N
[0094] A mixture of 7 (27.8 mg, 0.08 mmol ), 3-trifluoromethyl-phenylamine
(21.4 mg, 0.098
mmol), HATU (42 mg, 0.11 mmol), and diisopropylethyl amine (41 L, 0.24 mmol)
in DMF (1.5
mL) is kept stirring for 15 hours. The reaction mixture is concentrated and
purified by Prep-
HPLC to afford 8. MS (ESI) m/z: 469.2 (M+1)+.
Example 3
4-Methyl-3-(3-methyl-l-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)-N-(3-(4-
methylpiperazin-l-yl)-5-(trifluoromethyl)phenyl)benzamide (11)
N^N
I
NH NH N'CN 'N ANHNH2 N^N NH2
2CN Boc `N
Et0 Me EtOH, 40 oC EtO Me Benzene, reflux
Boc N~
9 10
F F
F
H
1. I N
C Pd(OAc)2, Cs2CO3 F F
0 ON F
H
NN HN N 4
4,5-Bis(diphenylphosphine)-9,9-dimethylxanthane N't~'A N~\N O ON
H 2. TFA:DCM/1:1 11
(E)-ethyl N-cyanoacetimidate (9)
N,CN
I
EtO Me
33
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[0095] A solution of acetimidic acid ethyl ester (5.0g, 40.4 mmol) and
cyanamide (8.6 g,
205.1 mmol) in ethanol (60 mL) is heated at 40 C under nitrogen for 2 hrs.
The ammonium
chloride formed during the reaction is filtered off, and the filtrate is
concentrated in vacuo to
afford crude product. The crude product is dissolved in ethyl acetate and
washed with water and
brine. The combined organic layers are dried with MgSO4 and concentrated in
vacuo to give (E)-
ethyl N-cyanoacetimidate as a white solid. 'H NMR (400 MHz, CD3OD) 8 4.14 (q,
J = 7.0 Hz,
2H), 1.98 (s, 3H), 1.23 (t, J= 7.0 Hz, 3H).
6-(5-Amino-3-methyl-[1,2,4]triazol-1-yl)-pyrimidin-4-yll-methyl-carbamic acid
tert-
butyl ester (10)
N^N NH2
)'N
Boc ,N
[0096] A mixture of (E)-ethyl N-cyanoacetimidate (1.2g, 10.7 mmol) and tert-
butyl 6-
hydrazinylpyrimidin-4-yl (methyl)carbamate (1.7 g, 7.13 mmol) in toluene (12
mL) is refluxed
overnight. The reaction mixture is purified on chromatography (silica gel, 10-
50%
EtOAc/Hexane) to give the desired product as a white solid. 1H NMR (400 MHz,
CD3OD) 8 8.70
(d, J= 0.8 Hz, 1H), 8.44 (d, J= 0.8 Hz, 1H), 3.44 (s, 3H), 2.38 (s, 3H), 1.53
(s, 9H). MS (ESI)
m/z: 306.1 (M+H)+.
4-Methyl-3-(3-methyl-l-(6-(methylamino)pyrimidin-4-yl)-1H-1,2,4-triazol-5-
ylamino)-
N-(3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)benzamide (11)
F F
F
N C
N^N HN
\N~N),-- N O CN ~
H , N ~ \
[0097] A mixture of compound 10 (46.3mg, 0.15 mmol), 3-iodo-4-methyl-N-(3-(4-
methylpiperazin-1-yl)-5-(trifluoromethyl)phenyl)benzamide (84.0 mg, 0.167
mmol), Pd2(dba)3
(5.0 mg, 0.005 mmol), Xantphos (16.7 mg, 0.029 mmol) and CszCO3 (100 mg, 0.31
mmol) in
dioxane (4.OmL) is heated to 150 C for 20 min under microwave condition. The
resulting
reaction mixture is purified by HPLC to afford product. 1H NMR (400 MHz,
CD3OD) 8 8.49 (s,
34
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1H), 8.34 (s, 1H), 7.76 (s, 1H), 7.48-7.46 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H),
6.99 (s, 1H), 6.69 (s,
1H), 3.50-3.28 (m, 8H), 2.88 (s, 3H), 2.86 (s, 3H), 2.40 (s, 3H), 2.26 (s,
3H). MS (ESI) m/z:
581.2 (M+H)+.
Example 4
1- { 4-Methyl-3- [2-(6-methylamino-pyrimidin-4-yl)-2H- [ 1,2,4ltriazol-3-
ylaminol-phenyl} -3-(3-
pyrrolidin-1-_ l~yl-5-trifluoromethyl-phenyl)-urea (12)
F F
H2N ~ F
/ F F
o F
N~N HN \ NH2 N I~ -
~
~ / ^ I~
Hi" v_N ~ v N ~ H H ~
HN^%~N ~N N/~I
Triphosgene, I N=/ ~J
4 EtN(iso-Pr)2, CH2CI2 12
[0098] A solution of 4 (29.6 mg, 0.1 mmol) and diisopropylethyl amine (38 L,
0.22 mmol)
in 2 mL CH2C12 is added dropwise to a CH2C12 (1 mL) solution of triphosgene
(11 mg, 0.37
mmol) under N2. The mixture is stirred at room temperature for 15 min. This
solution is added
dropwise to a solution of 3-pyrrolidin-1-ylmethyl-5-trifluoromethyl-
phenylamine (27 mg, 0.11
mmol) and diisopropanylethylamine (32 L, 0.18 mmol) in CHZC12 (2 mL) over 2
min. The
mixture is stirred at rt for 30 min. The solvent is removed in vacuo and the
crude product is
purified by HPLC to afford compound 12. LC-MS (ESI) m/z: 567.2 (M+H)+.
[0099] Table 1 describes representative compounds obtained by the above
Examples.
Table 1
Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
1H NMR (400 MHz, DMSO) 8 8.75 (s, 1H),
~ o F F 8.55 (s, 1H), 8.50 (s, 1H), 8.15 (s, 1H), 8.10
13 N'`N HN ~ I H (d, 1H), 7.95 (s, 1H), 7.90 (s, 1H), 7.80 (d,
HN~J`N'~N 1H), 7.66 (t, 1H), 7.45 (d, 1H), 7.20 (d, 1H),
I NJ 6.85 (s, 1H), 2.90 (s, 3H), 2.50 (s, 3H), 2.05
(t, 3H). MS (ESI) m/z: 465.1(M+H)+.
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
1H NMR (400 MHz, DMSO) 8 8.75 (m, 2H),
~ 0 8.70 (s, 1H), 7.94 (s, 1H), 7.90 (s, 1H), 7.75
N^N HN N \~N (d, 1H), 7.40 (d, 1H), 7.25 (d, 1H), 6.85 (s,
14 HN~~`NN " ' 1H), 2.95 (s, 3H), 2.35 (s, 3H), 1.40 (s, 6H).
MS (ESI) m/z: 458.20(M+H)+
1H NMR (400 MHz, DMSO) 8 8.70 (m, 2H),
~ o OH 8.65 (s, 1H), 8.25 (s, 1H), 8.00 (s, 1H), 7.95
N^N HN N \~N (s, 1H), 7.80 (s, 1H), 7.45 (d, 1H), 7.25 (d,
15 HN~~`NN " 1H), 6.85 (s, 1H), 2.90 (s, 3H), 2.38 (s, 3H),
1.50 (s, 3H). MS (ESI) m/z: 460.1 (M+H)+
1H NMR (400 MHz, DMSO) 8 8.88 (d, 1H),
p F F 8.75 (s, 1H), 8.55 (s, 1H), 8.20 (s, 1H), 8.05
16 N N "N I H N (d, 1H), 7.95 (s, 1H), 7.90 (s, 1H), 7.45 (d,
A ~ 1H), 7.25 (d, 1H), 6.85 (s, 1H), 2.92 (s, 3H),
H I N-i 2.45 (s, 3H), 2.05 (t, 3H). MS (ESI) m/z:
466.10 (M+H)+
1H NMR (400 MHz, DMSO) 8 9.65 (s, 1H),
0 F 8.70 (s, 1H), 8.65 (s, 1H), 7.95 (s, 1H), 7.90
~ (s, 1H), 7.80 (s, 1H), 7.78 (s, 1H), 7.50 (s,
17 N HN ~ H 1H), 7.40 (d, 1H), 7.20 (d, 1H), 6.85 (s, 1H),
H jNN N 4.10 (m, 2H), 3.60 (m, 2H), 3.25 (m, 2H),
3.12 (m, 4H), 2.92 (s, 3H), 2.35 (s, 3H), 1.25
N
~ (t, 3H). MS (ESI) m/z: 581.2 (M+H)+
1H NMR (400 MHz, DMSO) 8 8.72 (s, 1H),
~ ~ 8.55 (s, 1H), 7.95 (s, 1H), 7.90 (s, 1H), 7.50
18 H \ (m, 2H), 7.40 (m, 2H), 7.20 (d, 1H), 7.10 (d,
HN N~N 1H), 6.85 (s, 1H), 4.70 (m, 1H), 2.90 (s, 3H),
2.35 (s, 3H), 1.30 (d, 6H). MS (ESI) nVz:
459.2 (M+H)+
1H NMR (400 MHz, DMSO) 8 8.75 (s, 1H),
' ~ ~F 8.55 (s, 1H), 8.00 (d, 1H), 7.90 (s, 1H), 7.85
19 N~J. ~\ " F (s, 1H), 7.70 (t, 2H), 7.60 (m, 1H), 7.40 (d,
"NNJ 1H), 7.25 (d, 1H), 6.85 (s, 1H), 6.65 (s, 1H),
2.88 (s, 3H), 2.35 (s, 3H). MS (ESI) m/z:
485.20 (M+H)+
36
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
1H NMR (400 MHz, DMSO) 8 8.72 (s, 1H),
8.55 (s, 1H), 8.00 (s, 1H), 7.95 (d, 1H), 7.90
20 NN HN N o CN (s, 1H), 7.70 (d, 1H), 7.60 (t, 1H), 7.46 (m,
HNA'ANJN H 1H), 7.40 (m, 1H), 7.22 (d, 1H), 6.85 (s, 1H),
N~ 2.90 (s, 3H), 2.38 (s, 3H), 2.05 (m, 2H), 1.70
(s, 3H), 0.86 (t, 3H). MS (ESI) m/z: 482.2
(M+H)+
1H NMR (400 MHz, DMSO) 8 8.72 (s, 1H),
8.55 (s, 1H), 8.05 (s, 1H), 7.95 (d, 1H), 7.90
N^N HN I N eCN (s, 1H), 7.75 (d, 1H), 7.60 (t, 1H), 7.40 (d,
21 HNA"J`NJ`N H 1H), 7.22 (d, 1H), 6.85 (s, 1H), 2.90 (s, 3H),
2.36 (s, 3H), 1.78 (s, 6H). MS (ESI) m/z:
468.20 M+H)+
1H NMR (400 MHz, DMSO) 8 8.70 (s, 1H),
o oH 8.55 (s, 1H), 8.05 (s, 1H), 7.95 (s, 1H), 7.90
N^N HN I N (s, 1H), 7.80 (d, 1H), 7.78 (d, 1H), 7.45 (d,
22 H
HNNN 1H), 7.40 (d, 1H), 7.20 (d, 1H), 6.85 (s, 1H),
2.90 (s, 3H), 2.36 (s, 3H), 1.50 (s, 6H). MS
(ESI) m/z: 459.20 (M+H)+
MS (ESI) m/z: 567.2 (M+H)+
F F
~ I
F N 23 NN HN \ H O
HN v N N N
I NJ ~~
MS (ESI) m/z: 553.2 (M+H)+
F F
/ I
F
24 iN HN \ H O~
HN v N N N
N~ ~~
H
MS (ESI) m/z: 582.2 (M+H)+
F F
O
F
~N HN
Nv ~
HN NN H 0
N~ -CN-
37
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 597.2 (M+H)+
F F
F
26 ~N/~N HN \/ IH H HN" v -N~N N
N~ ON
11-1
OH
MS (ESI) m/z: 568.2 (M+H)+
F F
~ I O
F
27 N~N HN \H HN~N~N
NJ O-CNH
F MS (ESI) m/z: 530.3(M+H)+
_ F
N~N HN H O~ ~
28 I/~
HN" v N , N
N=~
HO
HO
MS (ESI) m/z: 487.0 (M+H)+
F F
~
_ F
29 ~NN HN \ HO~/ F
HN v N N
MS (ESI) m/z: 487.1 (M+H)+
F F
F
30 N~N HN / \ H
HNAv N `'N F
MS (ESI) m/z: 503.1 (M+H)+
F
~
F
\
31 ~~ ~ HO( ci
HN N N
38
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 567.2 (M+H)+
F F
H F
32 ~NN HN \ N /
HN v N`'N C 0N N=/
MS (ESI) m/z: 457.20 (M+H)+
H
33 I^/~N HN N /
HN v N~N C
MS (ESI) m/z: 593.2 (M+H)+
F F
34 N N HN : N
H
/ ~ F
H~~v N`'N C N
N=~
N
MS (ESI) m/z: 607.2 (M+H)+
F F
NN HN : I
H
\ F
i /
35 N (
~N \N O N
N=~
N
MS (ESI) m/z: 581.2 (M+H)+
F F
H F
36 ~NN HN /
\ N
Hi" v _N JN 0 N
ON
MS (ESI) m/z: 553.2 (M+H)+
F F
37 NN HN \ N /
~H F
HZNN `'N O N
N=~
N
\
39
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 583.10 (M+H)+
F F
~
38 N HN \ N
H F
I /
HN v N11 N C N
iC NJ
N
MS (ESI) m/z: 597.20 (M+H)+
F F
~i N HN \
/ ~N
H \
F
/
39
HN" v N- `'N C N
iC NJ
N
MS (ESI) m/z: 567.2 (M+H)+
F F
N
40 N HN \ N /
H \ F
HZN~N'~N O N
N=~
N
/
MS (ESI) m/z: 621.3(M+H)+
FF
/ ~~H F
41 ~NN HN ~ N ~~
HN v N`'N C N
~ NJ ~~
N
MS (ESI) m/z: 595.20 (M+H)+
FF
42 ~NN HN :N (
/~ ~H F
HN v N`'N C N
I NJ ~~
N
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 611(M+H)+
FF
43 NN HN \ N ~ /
~H F
HN N`N O N
/O N=~
N
MS (ESI) m/z: 491.1 (M+H)+
H \
44 N~N HN N ~~ N
HN" v -N- `'N O
~ N=l
MS (ESI) m/z: 470.1 (M+H)+
i
~ H N
45 NN HN \ N I /
HN v N`-N O F
I N~ FF
MS (ESI) m/z: 539.20 (M+H)+
/~ F
H
NI~NI HN \ N F
46 0 H
OH
MS (ESI) m/z: 500.2 (M+H)+
QO 47
H" N N H F
N=~ N
0
MS (ESI) m/z: 483.1 (M+H)+
H
/I
48 HNI \ N "
4g N~~N/II N O y
H
N~ CF3
41
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 582.2 (M+H)+
CF3
~^N HN H
49 N H l~-11NI
c ~
O
N p
H ~1MS (ESI) m/z: 479.2 (M+H)+
^N HN H ~
50 /
H
N
MS (ESI) m/z: 552.2 (M+H)+
/ I F F
NI H~ \ H F
51 H N N
N=~
MS (ESI) m/z: 541.3 (M+H)+
/I
H
c/~' N~~NI HN1 \ N I ~
52 vN~/~N~II N~\\N 0 iN
H N-l
MS (ESI) m/z: 526.2 (M+H)+
F F
~lN HNf N F
N"QN
53 N,/, N~ -%11
H NJ
MS (ESI) m/z: 515.2 (M+H)+
/I
H
NI~N HNI \ N
54 NN~~N/~N O iN
H N-/
MS (ESI) m/z: 595.2 (M+H)+
F F
H ~ F
55 H ~~NN,O N \ /
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 584.3 (M+H)+
H
/I
56 I N^/~N ~NHO N N
iN_) H N~
MS (ESI) m/z: 582.2 (M+H)+
F F
H F
I~
57 O
NH ~N N N \ /
MS (ESI) m/z: 571.3 (M+H)+
N~N HN N \
58 NMH~ NJN O N
MS (ESI) m/z: 552.2 (M+H)+
F F
I
H F
\ N
N N O ~
59 N~
H N~
MS (ESI) m/z: 495.2 (M+H)+
F F
/ I
H F
60 /~ N^N HNI \ N
~\NN O
H
MS (ESI) m/z: 484.2 (M+H)+
H
NII^N HN \ /IN ~
61 2~11 N , NN O I ~ N
H N-/
MS (ESI) m/z: 513.2 (M+H)+
F F
H F
N N HN N \ /
62 /O~/~N/\~N11QN O
H NJ
MS (ESI) m/z: 502.2 (M+H)+
H
/I
~N HN \ N I \
iN
63 /O~\NN~N 0
H N-/
43
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 458.2 (M+H)+
H
NN HN \ N
64 H iNA\ N O N
MS (ESI) m/z: 470.1 (M+H)+
NN N \ CF3
65 Hi" v -N'\\N O N
MS (ESI) m/z: 553.2 (M+H)+
O
N~N HN \ I N CF3
66 \ ~ H
HN_ v _NI
N
CN N~/
NJl
MS (ESI) m/z: 542.3 (M+H)+
H
N_~NI HN \ N
67 HNI~NA O I iN
CNJ N~N
N
MS (ESI) m/z: 568.2 (M+H)+
~I O
\ N HN \ H I\ CF3
68 HNN\
N /
J N~/
COl
MS (ESI) m/z: 557.3 (M+H)+
~I ~~
NN HN \ N~y \Y ~
69 HN ~/ `N'~~ O ll\ii IN
? NJN
COl
44
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 554.2 (M+H)+
H
N-;~N N CF3
70 HN" v-NAN O
CN` N~/
NJl
MS (ESI) m/z: 569.2 (M+H)+
/
N~N \ I N CF3
71 HN' v NA O N/
? NJN
COJ
MS (ESI) m/z: 553.2 (M+H)+
H
N~-N N \ CF3
72 HN v N'~~ O I/
i i N
CN N~/
NJl
MS (ESI) m/z: 568.2 (M+H)+
H
NN HN/
\ N CF3
\ I 1
73 HN~ v N'~~ O
? NJN
COJ
MS (ESI) m/z: 571.2 (M+H)+
/ O
N~N HN \ I N CF3
74 H
HN ~NN F
CN N~/
NJl
CA 02690653 2009-12-14
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 571.2 (M+H)+
/ o
N~N HN \ I N \ CF3
~
75 H
HN N N /
CNJ N~ F
N
MS (ESI) m/z: 571.2 (M+H)+
/ o
NN HN \ I NCF3
76 H
HN \AN N F
CN N
NJl
MS (ESI) m/z: 587.2 (M+H)+
/ o
N~N HN \ I NCF3
77 H
~~
HN \ N N / CI
CN` N~/
NJl
MS (ESI) m/z: 542.3 (M+H)+
/ I o
N~-N HN H
78 HNN \N ~N
CN` N~/
NJl
MS (ESI) m/z: 550.2 (M+H)+
F
/ I V~N
N/ N HN \ H \ F
79 HNN~N CN` N~/
NJl
46
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 552.3 (M+H)+
O
\ ~ HN H CN
80 HNNA\N
CN` N~/
NJl
MS (ESI) m/z: 557.3 (M+H)+
/ ~ O
NN HN \ H OMe
\~
81 HN" v -NAN
CN` N~/
NJl
MS (ESI) m/z: 586.2 (M+H)+
/ O
82 ~ HN \ I H CF3
N F
COJ
MS (ESI) m/z: 586.2 (M+H)+
/ O
\ N HN \ I H N CF3
83 HN~N\
N,/N F
CO
MS (ESI) m/z: 586.2 (M+H)+
/I O
\ N HN \ H CF3
84 HNN'\\N F
J N~/
COl
47
CA 02690653 2009-12-14
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 602.2 (M+H)+
/I O
\ N HN \ H CF3
85 HNN'\\N / CI
J N~/
COl
MS (ESI) m/z: 557.3 (M+H)+
O
\ N HN \ H
86 HNNN N
J N ~/
COJ
MS (ESI) m/z: 565.2 (M+H)+
O I
\ i HN \ H \ F F
87 HNNN N
J N ~/
Co~
MS (ESI) m/z: 567.3 (M+H)+
/ ~ O
N
N N HfN \ H ~VC
88 HN v NN J N
a/
Co~
MS (ESI) m/z: 572.3 (M+H)+
/ ~ O
HN \ H OMe
89 HN ~NN
IJ N~/
COJ
48
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 680.4 (M+H)+
o
\ N HN H I\ CF3
90 HNN\N
J N'/
Nr (N)
Co~
MS (ESI) m/z: 752.4 (M+H)+
o
N HN H I\ CF3
N~
91 J\\AN\
f'Ji Na/N /
N CN'
C O-)-O+
MS (ESI) m/z: 540.2 (M+H)+
/ O
N~N HN \ I NCF3
H I
92 HN \ N\
N /
CN` N~/
OJl
MS (ESI) m/z: 540.2 (M+H)+
H
NN /
\ N \ CF3
93 HN N '\\ O I/
i i N
CN N~/
OJl
MS (ESI) m/z: 581.3 (M+H)+
o
\l N HN H CF3
94 HN/NN
J Na/
CJ
N
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 581.3 (M+H)+
/
H
Ni N \ N \ CF3
95 HN~ v N'\\ O I
NJN
CJ
N
MS (ESI) m/z: 652.3 (M+H)+
O
\ N HN H I\ CF3
96 HNN\N
J N~/
Nr (N)
CO H
MS (ESI) m/z: 602.2 (M+H)+
ci
/I /I
9J I_ II HfN \ H H\ CF3
HNN\N
CN\ N~/
NJl
F MS (ESI) m/z: 586.2 (M+H)+
\ N HN \ I H~H \ I CF3
98 HN~N\\N
CN` N~/
NJl
MS (ESI) m/z: 602.2 (M+H)+
/
H
NN HN \ N \ CF3
99 HN v N'~~N O I/ CI
J N~/
COJ
CA 02690653 2009-12-14
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 586.2 (M+H)+
~
NN HN ~ I N CF3
100 HN" v-N'\\N O I~ F
J N~/
COl
MS (ESI) m/z: 498.2 (M+H)+
~ O
N~N HN \ I N OF3
A H
101 HN ~ N/\\N
N~/
H IN,~
MS (ESI) m/z: 484.1 (M+H)+
~ I
\
^N HN\ ~ IN!N\ CF3
102 HNN'\\N
~ N
F MS (ESI) m/z: 502.1 (M+H)+
/ ~ O
103 ~ ~ H~H b CF3
Hi N \N
F MS (ESI) m/z: 502.1 (M+H)+
N^~ HN \ IH!H \ ICF3
104 HN v N\N
~ N /
MS (ESI) m/z: 518.1 (M+H)+
ci
N N ~ I I \~ IF3
C
õIl \ H H
105 HN \/`N \N
~ N /
51
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 596.3 (M+H)+
106 (NN)
a ~
~ õI1 HN H H CF3
Hi /\N\N
N~/
MS (ESI) m/z: 596.3 (M+H)+
/ 0 / ~~N-
107 HN" N~ \ I HN \ I CF3
NyN
MS (ESI) m/z: 556.2 (M+H)+
/
H
NN HN \ N
108 H CF3
I~
i~IINA O
N N
MS (ESI) m/z: 570.3 (M+H)+
N~N HN \ I N CF3
N I/ I
109 HN" v N'kO
MS (ESI) m/z: 596.2 (M+H)+
/
N^N HN \ I N \ CF3
110 H N N '\~N O I/ 0
N~ N~3
MS (ESI) m/z: 582.3 (M+H)+
/
H
N^N HN \ NCF3
111 HN" v~NA N O
No
N~ O~\
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 584.3 (M+H)+
H
N^N HN \ N \ CF3
112 HN v NA N C
NJ N
F MS (ESI) m/z: 502.2 (M+H)+
N~~~ HN \ I H~H \ I CF3
113 HN' 'NVN
~ N~/
ci MS (ESI) m/z: 518.1 (M+H)+
N\~~ HN \ I H~H \ I CF3
114 HN' v `N\N
~ N~/
MS (ESI) m/z: 596.3 (M+H)+
115 (NN)
W~N / I
õIl H1N \ H H \ CF3
HN /`N'\~
N~N
MS (ESI) m/z: 584.3 (M+H)+
N
116 Nf\
/ I
õIl HN \ H H CF3
H i / \ N \N
N ~/
fo- MS (ESI) m/z: 571.2 (M+H)+
1~N
117 ~ o ~
õIl HN H H \ CF3
H / \N~~N
N~/
53
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Compound Structure Physical data (1H NMR (400 MHz, DMSO),
MS (ESI) m/z)
MS (ESI) m/z: 571.2 (M+H)+
N
118 Of\
a / I
~/~HN H H \ CF3
H ~ N ,N
N~V
MS (ESI) m/z: 610.3 (M+H)+
NJ
119 ~ O
N ~N HN \ H H\ CF3
Hi" N
N
MS (ESI) m/z: 526.2 (M+H)+
Nrl~N HN N CF3
120
HN" v N/\\ O
NJN
MS (ESI) m/z: 553.5 (M+H)+
~
N HN \ I N OF3
121 HN N\\ H
, I N
N
Na
OH
Assays
[00100] Compounds of the present invention may be assayed to measure their
capacity to
inhibit a kinase panel, including but not limited to Alk, Abl, Aurora-A, B-
Raf, C-Raf, Bcr-Abl,
BRK, Blk, Bmx, BTK, C-Kit, C-Src, EphBl, EphB2, EphB4, FLT1, Fms, F1t3, Fyn,
FRK3,
JAK2, KDR, Lck, Lyn, PDGFRa, PDGFR(3, PKCa, p38 (p38 MAP kinase, SAPK2a), Src,
SIK,
Syk, Tie2 and TrkB kinases.
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B-Raf (Enzymatic assay)
[00101] Compounds of the invention may be tested for their ability to inhibit
the activity of B-
Raf. The assay is carried out in 384-well MaxiSorp plates (NUNC) with black
walls and clear
bottom. The substrate, IxBa is diluted in DPBS (1:750) and 15 l is added to
each well. The
plates are incubated at 4 C overnight and washed 3 times with TBST (25 mM
Tris, pH 8.0, 150
mM NaC1 and 0.05% Tween-20) using the EMBLA plate washer. Plates are blocked
by
Superblock (15 l/well) for 3 hours at room temperature, washed 3 times with
TBST and pat-
dried. Assay buffer containing 20 M ATP (10 l) is added to each well
followed by 100 nl or
500 nl of compound. B-Raf is diluted in the assay buffer (1 l into 25 l) and
10 l of diluted b-
Raf is added to each well (0.4 g/well). The plates are incubated at room
temperature for 2.5
hours. The kinase reaction is stopped by washing the plates 6 times with TBST.
Phosph-
IxBa (Ser32/36) antibody is diluted in Superblock (1:10,000) and 15 l is
added to each well.
The plates are incubated at 4 C overnight and washed 6 times with TBST. AP-
conjugated goat-
anti-mouse IgG is diluted in Superblock (1:1,500) and 15 l is added to each
well. Plates are
incubated at room temperature for 1 hour and washed 6 times with TBST. 15 l
of fluorescent
Attophos AP substrate (Promega) is added to each well and plates are incubated
at room
temperature for 15 minutes. Plates are read on Acquest or Analyst GT using a
Fluorescence
Intensity Program (Excitation 455 nm, Emission 580 nm).
B-Raf (Cellular Assay)
[0100] Compounds of the invention are tested in A375 cells for their ability
to inhibit
phosphorylation of MEK. A375 cell line (ATCC) is derived from a human melanoma
patient and
has a V599E mutation on the B-Raf gene. The levels of phosphorylated MEK are
elevated due to
the mutation of B-Raf. Sub-confluent to confluent A375 cells are incubated
with compounds for
2 hours at 37 C in serum free medium. Cells are then washed once with cold
PBS and lysed with
the lysis buffer containing 1 Io Triton X100. After centrifugation, the
supernatants are subjected
to SDS-PAGE, and then transferred to nitrocellulose membranes. The membranes
are then
subjected to western blotting with anti-phospho-MEK antibody (ser2l7/221)
(Cell Signaling).
The amount of phosphorylated MEK is monitored by the density of phospho-MEK
bands on the
nitrocellulose membranes.
Inhibition of cellular Bcr-Abl dependent proliferation (High Throughput
method)
CA 02690653 2009-12-14
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[0101] The murine cell line 32D hemopoietic progenitor cell line may be
transformed with
Bcr-Abl cDNA (32D-p210). These cells are maintained in RPMI/10 Io fetal calf
serum
(RPMI/FCS) supplemented with penicillin 50 g/mL, streptomycin 50 g/mL and L-
glutamine
200 mM. Untransformed 32D cells are similarly maintained with the addition of
15% of WEHI
conditioned medium as a source of IL3.
[0102] 50 l of a 32D or 32D-p210 cells suspension are plated in Greiner 384
well
microplates (black) at a density of 5000 cells per well. 50 nl of test
compound (1 mM in DMSO
stock solution) is added to each well (ST1571 is included as a positive
control). The cells are
incubated for 72 hours at 37 C, 5% COZ. 10 l of a 60% Alamar Blue solution
(Tek diagnostics)
is added to each well and the cells are incubated for an additiona124 hours.
The fluorescence
intensity (Excitation at 530 nm, Emission at 580 nm) is quantified using the
AcquestTM system
(Molecular Devices).
Inhibition of cellular Bcr-Abl dependent proliferation
[0103] 32D-p210 cells are plated into 96 well TC plates at a density of 15,000
cells per well.
50 L of two fold serial dilutions of the test compound (C,,,aX is 40 M) are
added to each well
(ST1571 is included as a positive control). After incubating the cells for 48
hours at 37 C, 5%
C02, 15 L of MTT (Promega) is added to each well and the cells are incubated
for an additional
hours. The optical density at 570 nm is quantified spectrophotometrically and
IC50 values, the
concentration of compound required for 50% inhibition, determined from a dose
response curve.
Effect on cell cycle distribution
[0104] 32D and 32D-p210 cells are plated into 96 well TC plates at 2.5x 106
cells per well in
5 ml of medium and test compound at 1 or 10 M is added (ST1571 is included as
a control).
The cells are then incubated for 24 or 48 hours at 37 C, 5% COZ. 2 ml of cell
suspension is
washed with PBS, fixed in 70% EtOH for 1 hour and treated with PBS/EDTA/RNase
A for 30
minutes. Propidium iodide (Cf=10 g/ml) is added and the fluorescence
intensity is quantified
by flow cytometry on the FACScaliburTM system (BD Biosciences). In some
embodiments, test
compounds of the present invention may demonstrate an apoptotic effect on the
32D-p210 cells
but not induce apoptosis in the 32D parental cells.
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Effect on Cellular Bcr-Abl Autophospho . lr a~
[0105] Bcr-Abl autophosphorylation is quantified with capture Elisa using a c-
Abl specific
capture antibody and an antiphosphotyrosine antibody. 32D-p210 cells are
plated in 96 well TC
plates at 2x105 cells per well in 50 L of medium. 50 L of two fold serial
dilutions of test
compounds (C,,,.,x is 10 M) are added to each well (ST1571 is included as a
positive control).
The cells are incubated for 90 minutes at 37 C, 5% COZ. The cells are then
treated for 1 hour on
ice with 150 L of lysis buffer (50 mM Tris-HC1, pH 7.4, 150 mM NaC1, 5 mM
EDTA, 1 mM
EGTA and 1% NP-40) containing protease and phosphatase inhibitors. 50 L of
cell lysate is
added to 96 well optiplates previously coated with anti-Abl specific antibody
and blocked. The
plates are incubated for 4 hours at 4 C. After washing with TBS-Tween 20
buffer, 50 L of
alkaline-phosphatase conjugated anti-phosphotyrosine antibody is added and the
plate is further
incubated overnight at 4 C. After washing with TBS-Tween 20 buffer, 90 L of a
luminescent
substrate are added and the luminescence is quantified using the AcquestTM
system (Molecular
Devices). In some embodiments, test compounds of the invention may inhibit the
proliferation of
the Bcr-Abl expressing cells, inhibiting the cellular Bcr-Abl
autophosphorylation in a
dose-dependent manner.
Effect on proliferation of cells expressing mutant forms of Bcr-Abl
[0106] Compounds of the invention may be tested for their antiproliferative
effect on Ba/F3
cells expressing either wild type or the mutant forms of Bcr-Abl (G250E,
E255V, T3151, F317L,
M351T) that confers resistance or diminished sensitivity to STI571. The
antiproliferative effect
of these compounds on the mutant-Bcr-Abl expressing cells and on the non
transformed cells
may be tested at 10, 3.3, 1.1 and 0.37 M as described above (in media lacking
IL3). The IC50
values of the compounds lacking toxicity on the untransformed cells are
determined from the
dose response curves obtained as described above.
FGFR-3 (Enzymatic Assay)
[0107] Kinase activity assay with purified FGFR-3 (Upstate) is carried out in
a final volume
of 10 L containing 0.25 g/mL of enzyme in kinase buffer (30 mM Tris-HC1
pH7.5, 15 mM
MgC12, 4.5 mM MnC12, 15 M Na3VO4 and 50 g/mL BSA), and substrates (5 g/mL
biotin-
poly-EY(Glu, Tyr) (CIS-US, Inc.) and 3 M ATP). Two solutions are made: the
first solution of
l contains the FGFR-3 enzyme in kinase buffer was first dispensed into 384-
well format
57
CA 02690653 2009-12-14
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ProxiPlate (Perkin-Elmer) followed by adding 50 nL of compounds dissolved in
DMSO, then 5
l of second solution contains the substrate (poly-EY) and ATP in kinase buffer
was added to
each well. The reactions are incubated at room temperature for one hour,
stopped by adding 10
L of HTRF detection mixture, which contains 30 mM Tris-HC1 pH 7.5, 0.5 M KF,
50 mM
ETDA, 0.2 mg/mL BSA, 15 g/mL streptavidin-XL665 (CIS-US, Inc.) and 150 ng/mL
cryptate
conjugated anti-phosphotyrosine antibody (CIS-US, Inc.). After one hour of
room temperature
incubation to allow for streptavidin-biotin interaction, time resolved
florescent signals are read on
Analyst GT (Molecular Devices Corp.). IC50 values are calculated by linear
regression analysis
of the percentage inhibition of each compound at 12 concentrations (1:3
dilution from 50 M to
0.28 nM). In this assay, compounds of the invention have an IC50 in the range
of 10 nM to 2 M.
FGFR-3 (Cellular Assay)
[0108] Compounds of the invention are tested for their ability to inhibit
transformed Ba/F3-
TEL-FGFR-3 cells proliferation, which is dependent on FGFR-3 cellular kinase
activity. Ba/F3-
TEL-FGFR-3 are cultured up to 800,000 cells/mL in suspension, with RPMI 1640
supplemented
with 10% fetal bovine serum as the culture medium. Cells are dispensed into
384-well format
plate at 5000 cell/well in 50 L culture medium. Compounds of the invention
are dissolved and
diluted in dimethylsulfoxide (DMSO). Twelve points 1:3 serial dilutions are
made into DMSO to
create concentrations gradient ranging typically from 10 mM to 0.05 M. Cells
are added with
50 nL of diluted compounds and incubated for 48 hours in cell culture
incubator. AlamarBlue
(TREK Diagnostic Systems), which can be used to monitor the reducing
environment created by
proliferating cells, are added to cells at a final concentration of 10%. After
additional four hours
of incubation in a 37 C cell culture incubator, fluorescence signals from
reduced AlamarBlue
(Excitation at 530 nm, Emission at 580 nm) are quantified on Analyst GT
(Molecular Devices
Corp.). IC50 values are calculated by linear regression analysis of the
percentage inhibition of
each compound at 12 concentrations.
FLT3 and PDGFR(3
[0109] The effects of compounds of the invention on the cellular activity of
FLT3 and
PDGFRP may be conducted following identical methods as described above for
FGFR3 cellular
activity, using Ba/F3-FLT3-ITD and Ba/F3-Tel-PDGFR(3.
[0110] Compounds of the invention may be tested for their ability to inhibit
transformed
Ba/F3-FLT3-ITD or Ba/F3-Tel-PDGFRP cells proliferation, which is dependent on
FLT3 or
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PDGFR(3 cellular kinase activity. Ba/F3-FLT3-ITD or Ba/F3-Tel-PDGFRP are
cultured up to
800,000 cells/mL in suspension, with RPMI 1640 supplemented with 10% fetal
bovine serum as
the culture medium. Cells are dispensed into 384-well format plate at 5000
cell/well in 50 L
culture medium. Compounds of the invention are dissolved and diluted in
dimethylsulfoxide
(DMSO). Twelve points 1:3 serial dilutions are made into DMSO to create
concentrations
gradient ranging typically from 10 mM to 0.05 M. Cells are added with 50 nL
of diluted
compounds and incubated for 48 hours in cell culture incubator. AlamarBlue
(TREK
Diagnostic Systems), which can be used to monitor the reducing environment
created by
proliferating cells, are added to cells at final concentration of 10%. After
additional four hours of
incubation in a 37 C cell culture incubator, fluorescence signals from
reduced AlamarBlue
(Excitation at 530 nm, Emission at 580 nm) are quantified on Analyst GT
(Molecular Devices
Corp.). IC50 values are calculated by linear regression analysis of the
percentage inhibition of
each compound at 12 concentrations.
C-Kit
[0111] Compounds of the invention may be tested for inhibition of SCF
dependent
proliferation using Mo7e cells which endogenously express c-Kit in a 96-well
format. Briefly,
two-fold serially diluted test compounds (C,,,.,x = 10 M) are evaluated for
their antiproliferative
activity on Mo7e cells stimulated with human recombinant SCF. After 48 hour
incubation at 37
C, cell viability is measured by using a MTT colorimetric assay from Promega.
Upstate KinaseProfilerTm - Radio-enzymatic filter binding assay
[0112] Compounds of the invention may be assessed for their ability to inhibit
individual
member of a panel of kinases (a partial, non-limiting list of kinases
includes: Alk, Abl, Aurora-A,
B-Raf, Bcr-Abl, BRK, Blk, Bmx, C-Kit, C-Raf, C-Src, CSK, EphB, FLT1, Fms, Fyn,
JAK2,
KDR, Lck, Lyn, PDGFRa, PDGFR(3, PKCa, p38 (p38 MAP kinase, SAPK2a), SIK, Src,
Syk,
Tie2 and TrkB kinases). The compounds are tested in duplicates at a final
concentration of 10
M following this generic protocol, using varying kinase buffer composition and
substrates for
the different kinases included in the "Upstate KinaseProfilerTm panel. Kinase
buffer (2.5 L, lOx
- containing MnC12 when required), active kinase (0.001-0.01 Units; 2.5 L),
specific or
Poly(G1u4-Tyr) peptide (5-500 M or.01 mg/ml) in kinase buffer and kinase
buffer (50 M; 5
L) are mixed in an eppendorf on ice. A Mg/ATP mix (10 L; 67.5 (or 33.75) mM
MgC12, 450
59
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WO 2008/157131 PCT/US2008/066426
(or 225) M ATP and 1 Ci/ l [y-32P]-ATP (3000Ci/mmol)) is added and the
reaction is
incubated at about 30 C for about 10 minutes. The reaction mixture is spotted
(20 L) onto a
2cm x 2cm P81 (phosphocellulose, for positively charged peptide substrates) or
Whatman No. 1
(for Poly (G1u4-Tyr) peptide substrate) paper square. The assay squares are
washed 4 times, for
minutes each, with 0.75% phosphoric acid and washed once with acetone for 5
minutes. The
assay squares are transferred to a scintillation vial, 5 ml scintillation
cocktail are added and 32P
incorporation (cpm) to the peptide substrate is quantified with a Beckman
scintillation counter.
Percentage inhibition is calculated for each reaction.
[0113] Compounds of Formula (1), (2) or (3) in free form or in
pharmaceutically acceptable
salt form, may exhibit valuable pharmacological properties, for example, as
indicated by the in
vitro tests described in this application. The IC50 value in those experiments
is given as that
concentration of the test compound in question that results in a cell count
that is 50 % lower than
that obtained using the control without inhibitor. In general, compounds of
the invention have
IC50 values from 1 nM to 10 M against one or more of the following kinases:
Alk, Abl, Aurora-
A, B-Raf, C-Raf, Bcr-Abl, BRK, Blk, Bmx, BTK, C-Kit, C-Src, EphBl, EphB2,
EphB4, FLT1,
Fms, F1t3, Fyn, FRK3, JAK2, KDR, Lck, Lyn, PDGFRa, PDGFR(3, PKCa, p38 (p38 MAP
kinase, SAPK2a), Src, SIK, Syk, Tie2 and TrkB kinases.
[0114] In some examples, compounds of the invention have IC50 values from 0.01
M to 5
M. In other examples, compounds of the invention have IC50 values from 0.01 M
to 1 M, or
more particularly from 1 nM to 1 M. In yet other examples, compounds of the
invention have
IC50 values of < 0-100 nM, 100-250 nM, 250-500 nM, or > 500 nM. Compounds of
the
invention may also have IC50 values of less than 1 nM or more than 10 M.
[0115] Compounds of Formula (1), (2) or (3) may exhibit a percentage
inhibition of greater
than 50%, or in other embodiments, may exhibit a percentage inhibition greater
than about 70%,
against one or more of the following kinases at 10 M: Alk, Abl, Aurora-A, B-
Raf, C-Raf, Bcr-
Abl, BRK, Blk, Bmx, BTK, C-Kit, C-Src, EphBl, EphB2, EphB4, FLT1, Fms, F1t3,
Fyn, FRK3,
JAK2, KDR, Lck, Lyn, PDGFRa, PDGFR(3, PKCa, p38 (p38 MAP kinase, SAPK2a), Src,
SIK,
Syk, Tie2 and TrkB kinases.
[0116] It is understood that the examples and embodiments described herein are
for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of this
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application and scope of the appended claims. All publications, patents, and
patent applications
cited herein are hereby incorporated by reference for all purposes.
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